Novel lysophospholipid acyltransferase

ABSTRACT

The present invention provides novel lysophospholipid acyltransferases. The object of the present invention is attained by the nucleotide sequences of SEQ ID NOs: 1 and 6 and the amino acid sequences of SEQ ID NOs: 2 and 7 of the present invention.

This application is a Divisional of U.S. application Ser. No.13/255,390, which is the National Stage Application of InternationalApplication PCT/JP2010/055244, filed Mar. 25, 2010, which claimspriority to Japanese Application No. 2009-076809, filed Mar. 26, 2009.The disclosure of each of application Ser. No. 13/255,390 andPCT/JP2010/055244 are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 16, 2011, isnamed P40741.txt and is 64,982 bytes in size.

TECHNICAL FIELD

The present invention relates to novel lysophospholipidacyltransferases.

BACKGROUND ART

Biosynthesis of Polyunsaturated Fatty Acids

Fatty acids are major components of lipids such as phospholipids andtriacylglycerols. Fatty acids containing two or more unsaturated bondsare collectively referred to as polyunsaturated fatty acids (PUFAs), andare known to include arachidonic acid, dihomo-γ-linolenic acid,eicosapentaenoic acid, docosahexaenoic acid, etc. Various physiologicalactivities have been reported for these fatty acids (non-patent document1).

These polyunsaturated fatty acids are expected to find applications invarious fields, but some of them cannot be synthesized in vivo inanimals. This has led to development of methods for obtainingpolyunsaturated fatty acids by culturing various microorganisms.Attempts to produce polyunsaturated fatty acids in plants have also beenmade. In such cases, polyunsaturated fatty acids are known to beaccumulated as components of reserve lipids such as triacylglycerols,for example, in microbial cells or plant seeds.

Among the polyunsaturated fatty acids, arachidonic acid has attractedattention as an intermediate metabolite in the synthesis ofprostaglandins, leukotrienes and the like, and many attempts have beenmade to apply it as a material for functional foods and medicaments.Furthermore, arachidonic acid is contained in breast milk so that it isimportant for the growth of infants, especially for the growth of fetallength and brain, and therefore, it also attracts attention in anutritional aspect as a necessary component for the growth of infants aswell as DHA (docosahexaenoic acid).

Arachidonic acid is biosynthesized by the pathway shown in FIG. 1.Specifically, arachidonic acid is produced through several chainelongation and desaturation steps from palmitic acid generated by denovo fatty acid synthesis. In this pathway, an elongase and Δ9desaturase act on acyl-CoA. On the other hand, Δ12 desaturase, Δ6desaturase and Δ5 desaturase are known to act on the acyl groups ofphospholipids such as phosphatidylcholine (non-patent document 2). Thus,acyl transfer between acyl-CoA and phospholipids is required in thebiosynthesis of PUFAs such as arachidonic acid. Without being limited tothe biosynthesis of PUFAs, replacement of only fatty acids afterbiosynthesis of phospholipids is known as “remodeling” of phospholipids,and lysophospholipid acyltransferases (hereinafter referred to as“LPLATs”) are known to be involved in this reaction (non-patent document3).

Biosynthesis of Triacylglycerols

Among reserve lipids, triacylglycerols are synthesized in vivo asfollows. Glycerol-3-phosphate is acylated with glycerol-3-phosphateacyltransferase (hereinafter sometimes referred to as “GPAT”) at thehydroxyl group in the 1-position (α-position) to form lysophosphatidicacid (hereinafter sometimes referred to as “LPA”). LPA is alysophospholipid containing only one acyl group, and is acylated withlysophosphatidic acid acyltransferase (hereinafter sometimes referred toas “LPAAT”) to form phosphatidic acid (hereinafter sometimes referred toas “PA”). This PA is dephosphorylated by phosphatidic acid phosphataseto form diacylglycerol, which is in turn acylated with diacylglycerolacyltransferase (hereinafter sometimes referred to as “DGAT”) to formtriacylglycerol. Acyl-CoA: cholesterol acyltransferase (hereinaftersometimes referred to as “ACAT”) and lysophosphatidylcholineacyltransferase (hereinafter sometimes referred to as “LPCAT”) and thelike are known to be indirectly involved in the biosynthesis oftriacylglycerols.

Biosynthesis of Phospholipids

PA produced from LPA by the action of LPAAT as described above serves asa precursor in the biosynthesis of various phospholipids. For example,important phospholipids such as phosphatidylethanolamine (PE),phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol(PI), and phosphatidylglycerol (PG) are biosynthesized from PA. Thus, PAis not only an intermediate in lipid synthesis, but also anintracellular and intercellular lipid mediator having a very wide rangeof biological and pharmacological effects such as cell proliferation,platelet aggregation, smooth muscle contraction, promotion of cancerinvasion, etc.

Lysophospholipid Acyltransferases

As described above, LPLATs are believed to be involved in PUFAbiosynthesis. The LPLATs collectively refer to enzymes having theactivity of introducing an acyl group into lysophospholipids, andinclude those having various names based on the specificity for thesubstrate, i.e., the molecular species of the lysophospholipid used as asubstrate. One example is LPAAT that is involved in the synthesis oftriacylglycerols and phospholipids using LPA as a substrate. Otherlysophospholipids on which LPLATs act include lysophosphatidylcholine(LPC), lysophosphatidylserine (LPS), lysophosphatidylethanolamine (LPE),lysophosphatidylinositol (LPI), etc. Thus, the enzymes are called LPAAT,LPCAT, lysophosphatidylserine acyltransferase (LPSAT),lysophosphatidylinositol acyltransferase (LPIAT) and the like based onthe molecular species on which they act. Each enzyme may specificallyact on one lysophospholipid or multiple specific lysophospholipids. Forexample, LPLATs called as LPAAT include those acting on not only LPA butalso LPC, LPE, etc.

Sequence Profile-Based Classification of LysophospholipidAcyltransferases

LPLATs are classified as glycerophospholipid acyltransferases. Theglycerophospholipid acyltransferases are thought to fall into threegroups from amino acid sequence comparison, i.e., LPAAT family, MBOAT(membrane-bound O-acyltransferase) family and DGAT2 family (non-patentdocument 5). Enzymes belonging to the LPAAT family are commonlycharacterized by a membrane-bound domain and a sequentially conservedmotif (LPAAT motif). The enzymes belonging to the LPAAT family membersinclude LPAAT, GPAT, etc. Enzymes included in the MBOAT family arecommonly characterized by a membrane-bound domain. The MBOAT family isknown to include DGAT, ACAT and the like in addition to LPLAT. Inanimals or the like, some enzymes belonging to the MBOAT family arethought to be responsible for the remodeling reaction critical formembrane phospholipid synthesis.

LPLATs have been reported in a broad spectrum of organisms fromunicellular organisms such as bacteria and yeast to higher organismssuch as mammals. In yeast (Saccharomyces cerevisiae) belonging to fungi,SLC1 (YDL052C) and SLC4 (YOR175C) (herein sometimes referred to as“ALE1” or “LPT1”) are known as membrane-bound LPLAT genes (non-patentdocument 5). In animals, multiple LPLAT homologs are known to exist,including those responsible for the reaction of acting on LPA in the denovo triglyceride synthesis system to yield PA and those responsible forphospholipid remodeling (non-patent document 6).

In the lipid-producing fungus Mortierella alpina (hereinafter sometimesreferred to as “M. alpina”), four LPLATs have been obtained, all ofwhich belong to the LPAAT family (patent documents 1-3). However, noreport shows that any LPLAT belonging to the MBOAT family has beenobtained from M. alpina.

REFERENCES Patent Documents

-   Patent document 1: International Publication No. WO2004/087902-   Patent document 2: U.S. Patent Application Publication No.    US2006/0094090-   Patent document 3: International Publication No. WO2008/146745

Non-Patent Documents

-   Non-patent document 1: Lipids, 39, 1147 (2004)-   Non-patent document 2: J.B.C., 278(37), 35115-35126, (2003)-   Non-patent document 3: J.B.C., 276(29), 26745-26752, (2001)-   Non-patent document 4: Proc. Natl. Acad. Sci., 105(8), 2830-2835,    (2008)-   Non-patent document 5: J.B.C., 282(42), 30845-30855, (2007)-   Non-patent document 6: J.B.C., 284(1), 1-5, (2009)-   Non-patent document 7: Trends Biochem. Sci., 25, 111-112, (2000)-   Non-patent document 8: Journal of lipid research 2009 R80035    JLR200v1

SUMMARY OF INVENTION Technical Problems

As described above, phospholipid remodeling is essential in thebiosynthesis of PUFAs such as arachidonic acid, and LPLATs may beinvolved in this reaction. However, the LPAAT homologs hitherto knownhad the disadvantage that the proportion of PUFAs in total fatty acidscould not be sufficiently increased even if they were transferred andexpressed in host organisms. Therefore, there is a need to identifynovel nucleic acid and protein that would sufficiently increase theproportion of PUFAs in total fatty acids in a host when they aretransferred and expressed in the host. There is also a need to identifya nucleic acid and protein capable of producing fats with a high contentof industrially valuable fatty acids and to develop a method by whichvaluable fatty acids can be produced or the content of valuable fattyacids can be increased by using them.

Solution to Problems

An object of the present invention is to provide proteins and nucleicacids capable of producing valuable fats by expressing them in a hostcell to influence lipid metabolism of the host or to increase thecontent of a desired fatty acid.

In the biosynthesis of PUFAs such as arachidonic acid, phospholipidremodeling is essential. The lipid-producing fungus M. alpina canaccumulate large quantities of valuable PUFAs such as arachidonic acid,but any acyltransferase belonging to the MBOAT family involved in lipidremodeling as reported in animals or the like has not been obtained fromM. alpina. The inventor recognized this point and carefully studied toattain the above object, with the result that the inventor obtained cDNAencoding an enzyme belonging to the MBOAT family from M. alpina.Further, the inventor attempted to produce a fatty acid composition bytransforming the resulting cDNA into a highly proliferative host cellsuch as yeast to find that the host cell can produce a different fattyacid composition, especially a fatty acid composition having a highproportion of arachidonic acid as compared with fatty acid compositionsproduced by hosts transformed with vectors containing nucleic acidsencoding known LPAATs obtained from M. alpina. Thus, the inventorsucceeded in cloning genes for novel LPLATs different from known LPAATsand finally accomplished the present invention.

Accordingly, the present invention provides the following aspects.

(1) A nucleic acid of any one of (a)-(e) below:(a) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence with deletion, substitutionor addition of one or more amino acids in the amino acid sequence shownin SEQ ID NO: 2 or 7, and having lysophospholipid acyltransferaseactivity;(b) a nucleic acid that hybridizes under stringent conditions to anucleic acid consisting of a nucleotide sequence complementary to thenucleotide sequence consisting of SEQ ID NO: 1 or 6 and that comprises anucleotide sequence encoding a protein having lysophospholipidacyltransferase activity;(c) a nucleic acid that comprises a nucleotide sequence sharing anidentity of 80% or more with the nucleotide sequence consisting of SEQID NO: 1 or 6 and encoding a protein having lysophospholipidacyltransferase activity;(d) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence sharing an identity of 80%or more with the amino acid sequence consisting of SEQ ID NO: 2 or 7 andhaving lysophospholipid acyltransferase activity; and(e) a nucleic acid that hybridizes under stringent conditions to anucleic acid consisting of a nucleotide sequence complementary to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 or 7 and that comprises a nucleotidesequence encoding a protein having lysophospholipid acyltransferaseactivity.(2) The nucleic acid of (1), which is any one of (a)-(e) below:(a) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of a variant of the amino acid sequence shown in SEQID NO: 2 or 7 in which 1-50 amino acids are deleted, substituted oradded, and having lysophospholipid acyltransferase activity;(b) a nucleic acid that hybridizes under conditions of 2×SSC, 50° C. toa nucleic acid consisting of a nucleotide sequence complementary to thenucleotide sequence consisting of SEQ ID NO: 1 or 6 and that comprises anucleotide sequence encoding a protein having lysophospholipidacyltransferase activity;(c) a nucleic acid that comprises a nucleotide sequence sharing anidentity of 90% or more with the nucleotide sequence consisting of SEQID NO: 1 or 6 and encoding a protein having lysophospholipidacyltransferase activity;(d) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence sharing an identity of 90%or more with the amino acid sequence consisting of SEQ ID NO: 2 or 7 andhaving lysophospholipid acyltransferase activity; and(e) a nucleic acid that hybridizes under conditions of 2×SSC, 50° C. toa nucleic acid consisting of a nucleotide sequence complementary to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 or 7 and that comprises a nucleotidesequence encoding a protein having lysophospholipid acyltransferaseactivity.(3) A nucleic acid of any one of (a)-(e) below:(a) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence with deletion, substitutionor addition of one or more amino acids in the amino acid sequence shownin SEQ ID NO: 2 or 7, and having the activity of increasing theproportion of arachidonic acid in the compositional ratio of fatty acidsin a host transformed with a recombinant vector containing the nucleicacid as compared with the proportion in the compositional ratio of fattyacids in a host that has not been transformed with the vector;(b) a nucleic acid that hybridizes under stringent conditions to anucleic acid consisting of a nucleotide sequence complementary to thenucleotide sequence consisting of SEQ ID NO: 1 or 6 and that comprises anucleotide sequence encoding a protein having the activity of increasingthe proportion of arachidonic acid in the compositional ratio of fattyacids in a host transformed with a recombinant vector containing thenucleic acid as compared with the proportion in the compositional ratioof fatty acids in a host that has not been transformed with the vector;(c) a nucleic acid that comprises a nucleotide sequence sharing anidentity of 80% or more with the nucleotide sequence consisting of SEQID NO: 1 or 6 and encoding a protein having the activity of increasingthe proportion of arachidonic acid in the compositional ratio of fattyacids in a host transformed with a recombinant vector containing thenucleic acid as compared with the proportion in the compositional ratioof fatty acids in a host that has not been transformed with the vector;(d) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence sharing an identity of 80%or more with the amino acid sequence consisting of SEQ ID NO: 2 or 7 andhaving the activity of increasing the proportion of arachidonic acid inthe compositional ratio of fatty acids in a host transformed with arecombinant vector containing the nucleic acid as compared with theproportion in the compositional ratio of fatty acids in a host that hasnot been transformed with the vector; and(e) a nucleic acid that hybridizes under stringent conditions to anucleic acid consisting of a nucleotide sequence complementary to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 or 7 and that comprises a nucleotidesequence encoding a protein having the activity of increasing theproportion of arachidonic acid in the compositional ratio of fatty acidsin a host transformed with a recombinant vector containing the nucleicacid as compared with the proportion in the compositional ratio of fattyacids in a host that has not been transformed with the vector.(4) The nucleic acid of (3), which is any one of (a)-(e) below:(a) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence with deletion, substitutionor addition of 1-50 amino acids in the amino acid sequence shown in SEQID NO: 2 or 7, and having the activity of increasing the proportion ofarachidonic acid in the compositional ratio of fatty acids in a hosttransformed with a recombinant vector containing the nucleic acid ascompared with the proportion in the compositional ratio of fatty acidsin a host that has not been transformed with the vector;(b) a nucleic acid that hybridizes under conditions of 2×SSC, 50° C. toa nucleic acid consisting of a nucleotide sequence complementary to thenucleotide sequence consisting of SEQ ID NO: 1 or 6 and that comprises anucleotide sequence encoding a protein having the activity of increasingthe proportion of arachidonic acid in the compositional ratio of fattyacids in a host transformed with a recombinant vector containing thenucleic acid as compared with the proportion in the compositional ratioof fatty acids in a host that has not been transformed with the vector;(c) a nucleic acid that comprises a nucleotide sequence sharing anidentity of 90% or more with the nucleotide sequence consisting of SEQID NO: 1 or 6 and encoding a protein having the activity of increasingthe proportion of arachidonic acid in the compositional ratio of fattyacids in a host transformed with a recombinant vector containing thenucleic acid as compared with the proportion in the compositional ratioof fatty acids in a host that has not been transformed with the vector;(d) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence sharing an identity of 90%or more with the amino acid sequence consisting of SEQ ID NO: 2 or 7 andhaving the activity of increasing the proportion of arachidonic acid inthe compositional ratio of fatty acids in a host transformed with arecombinant vector containing the nucleic acid as compared with theproportion in the compositional ratio of fatty acids in a host that hasnot been transformed with the vector; and(e) a nucleic acid that hybridizes under conditions of 2×SSC, 50° C. toa nucleic acid consisting of a nucleotide sequence complementary to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 or 7 and that comprises a nucleotidesequence encoding a protein having the activity of increasing theproportion of arachidonic acid in the compositional ratio of fatty acidsin a host transformed with a recombinant vector containing the nucleicacid as compared with the proportion in the compositional ratio of fattyacids in a host that has not been transformed with the vector.(5) The nucleic acid of any one of (1)-(4) wherein the encoded proteinbelongs to the membrane-bound O-acyltransferase family.(6) A nucleic acid of any one of (a)-(d) below:(a) a nucleic acid that comprises the nucleotide sequence shown in SEQID NO: 1 or 6 or a partial sequence thereof;(b) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 7or a partial sequence thereof;(c) a nucleic acid that comprises the nucleotide sequence shown in SEQID NO: 4 or 9 or a partial sequence thereof; and(d) a nucleic acid that comprises the nucleotide sequence shown in SEQID NO: 5 or 10 or a partial sequence thereof.(7) A protein of (a) or (b) below:(a) a protein consisting of an amino acid sequence with deletion,substitution or addition of one or more amino acids in a variant of theamino acid sequence of SEQ ID NO: 2 or 7, and having lysophospholipidacyltransferase activity; or(b) a protein consisting of an amino acid sequence sharing an identityof 80% or more with the amino acid sequence consisting of SEQ ID NO: 2or 7 and having lysophospholipid acyltransferase activity.(8) The protein of (7), which is (a) or (b) below:(a) a protein consisting of an amino acid sequence with deletion,substitution or addition of 1-50 amino acids in the amino acid sequenceof SEQ ID NO: 2 or 7, and having lysophospholipid acyltransferaseactivity; or(b) a protein consisting of an amino acid sequence sharing an identityof having 90% or more with the amino acid sequence consisting of SEQ IDNO: 2 or 7 and having lysophospholipid acyltransferase activity.(9) A protein of (a) or (b) below:(a) a protein consisting of an amino acid sequence with deletion,substitution or addition of one or more amino acids in the amino acidsequence of SEQ ID NO: 2 or 7, and having the activity of increasing theproportion of arachidonic acid in the compositional ratio of fatty acidsin a host transformed with a recombinant vector containing a nucleicacid encoding the amino acid sequence as compared with the proportion inthe compositional ratio of fatty acids in a host that has not beentransformed with the vector; or(b) a protein consisting of an amino acid sequence sharing an identityof 80% or more with the amino acid sequence consisting of SEQ ID NO: 2or 7 and having the activity of increasing the proportion of arachidonicacid in the compositional ratio of fatty acids in a host transformedwith a recombinant vector containing a nucleic acid encoding the aminoacid sequence as compared with the proportion in the compositional ratioof fatty acids in a host that has not been transformed with the vector.(10) The protein of (9), which is (a) or (b) below:(a) a protein consisting of an amino acid sequence with deletion,substitution or addition of 1-50 amino acids in the amino acid sequenceof SEQ ID NO: 2 or 7, and having the activity of increasing theproportion of arachidonic acid in the compositional ratio of fatty acidsin a host transformed with a recombinant vector containing a nucleicacid encoding the amino acid sequence as compared with the proportion inthe compositional ratio of fatty acids in a host that has not beentransformed with the vector; or(b) a protein consisting of an amino acid sequence sharing an identityof 90% or more with the amino acid sequence consisting of SEQ ID NO: 2or 7 and having the activity of increasing the proportion of arachidonicacid in the compositional ratio of fatty acids in a host transformedwith a recombinant vector containing a nucleic acid encoding the aminoacid sequence as compared with the proportion in the compositional ratioof fatty acids in a host that has not been transformed with the vector.(11) The protein of any one of (7)-(10), which belongs to themembrane-bound O-acyltransferase family.(12) A protein consisting of the amino acid sequence shown in SEQ ID NO:2 or 7.(13) A recombinant vector containing the nucleic acid of any one of(1)-(6).(14) A cell transformed with the recombinant vector of (13).(15) A fatty acid composition obtained by culturing the transformed cellof (14) wherein the proportion of arachidonic acid in the compositionalratio of fatty acids in said fatty acid composition is higher than theproportion of arachidonic acid in the fatty acid composition obtained byculturing a non-transformed host.(16) A method for preparing a fatty acid composition, comprisingcollecting the fatty acid composition of (15) from cultures of thetransformed cell of (14).(17) A food product comprising the fatty acid composition of (15).(18) A method for using the recombinant vector of (13) to increase theproportion of arachidonic acid in the compositional ratio of fatty acidsin a host transformed with the vector as compared with the proportion incompositional ratio of fatty acids in a host that has not beentransformed with the vector.(19) A nucleic acid of any one of (a)-(e) below:(a) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence with deletion, substitutionor addition of one or more amino acids in the amino acid sequence shownin SEQ ID NO: 2 or 7, and involved in the conversion from 18:3(n-6)-PLto 18:3(n-6)-CoA and/or conversion from DGLA-CoA to DGLA-PL;(b) a nucleic acid that hybridizes under stringent conditions to anucleic acid consisting of a nucleotide sequence complementary to thenucleotide sequence consisting of SEQ ID NO: 1 or 6 and that comprises anucleotide sequence encoding a protein involved in the conversion from18:3(n-6)-PL to 18:3(n-6)-CoA and/or conversion from DGLA-CoA toDGLA-PL;(c) a nucleic acid that comprises a nucleotide sequence sharing anidentity of 80% or more with the nucleotide sequence consisting of SEQID NO: 1 or 6 and encoding a protein involved in the conversion from18:3(n-6)-PL to 18:3(n-6)-CoA and/or conversion from DGLA-CoA toDGLA-PL;(d) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence sharing an identity of 80%or more with the amino acid sequence consisting of SEQ ID NO: 2 or 7 andinvolved in the conversion from 18:3(n-6)-PL to 18:3(n-6)-CoA and/orconversion from DGLA-CoA to DGLA-PL; and(e) a nucleic acid that hybridizes under stringent conditions to anucleic acid consisting of a nucleotide sequence complementary to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 or 7 and that comprises a nucleotidesequence encoding a protein involved in the conversion from 18:3(n-6)-PLto 18:3(n-6)-CoA and/or conversion from DGLA-CoA to DGLA-PL.(20) A protein of (a) or (b) below:(a) a protein consisting of an amino acid sequence with deletion,substitution or addition of one or more amino acids in the amino acidsequence of SEQ ID NO: 2 or 7, and involved in the conversion from18:3(n-6)-PL to 18:3(n-6)-CoA and/or conversion from DGLA-CoA toDGLA-PL; or(b) a protein consisting of an amino acid sequence sharing an identityof 80% or more with the amino acid sequence consisting of SEQ ID NO: 2or 7 and involved in the conversion from 18:3(n-6)-PL to 18:3(n-6)-CoAand/or conversion from DGLA-CoA to DGLA-PL.

Advantageous Effects of Invention

The LPLATs of the present invention allows an improvement in the abilityto produce fatty acids, such as arachidonic acid, and/or reserve lipids,and hence is preferred as means for improving the productivity ofpolyunsaturated fatty acids in microorganisms and plants. Thus, they canprovide lipids having desired characteristics or effects so that theycan be usefully applied for use in foods, cosmetics, pharmaceuticals,soaps, etc.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing the biosynthetic pathway ofarachidonic acid. In FIG. 1, the abbreviations have the followingmeanings: PL, phospholipid; CoA, coenzyme A; DS, desaturase (fatty aciddesaturase enzyme); GLELO, fatty acid elongase; 18:0, stearoyl group;18:1, oleoyl group; 18:2, lilnoyl group; 18:3(n-6), γ-lilnoleyl group;DGLA, dihomo-γ-lilnoleyl group; ARA, arachidoyl group.

FIG. 2 shows the full-length cDNA sequence (SEQ ID NO: 4) of LPLAT5 fromM. alpina strain 1S-4 and the amino acid sequence (SEQ ID NO: 2) deducedtherefrom.

FIG. 3 shows the full-length cDNA sequence (SEQ ID NO: 9) of LPLAT6 fromM. alpina strain 1S-4 and the amino acid sequence (SEQ ID NO: 7) deducedtherefrom.

FIG. 4A shows a comparison between the genomic sequence (SEQ ID NO: 5)and the ORF sequence (SEQ ID NO: 1) of LPLAT5 from M. alpina strain1S-4.

FIG. 4B shows a comparison between the genomic sequence (SEQ ID NO: 5continued) and the ORF sequence (SEQ ID NO: 1 continued) of LPLAT5 fromM. alpina strain 1S-4.

FIG. 4C shows a comparison between the genomic sequence (SEQ ID NO: 5continued) and the ORF sequence (SEQ ID NO: 1 continued) of LPLAT5 fromM. alpina strain 1S-4.

FIG. 5A shows a comparison between the genomic sequence (SEQ ID NO: 10)and the ORF sequence (SEQ ID NO: 6) of LPLAT6 from M. alpina strain1S-4.

FIG. 5B shows a comparison between the genomic sequence (SEQ ID NO: 10continued) and the ORF sequence (SEQ ID NO: 6 continued) of LPLAT6 fromM. alpina strain 1S-4.

FIG. 5C shows a comparison between the genomic sequence (SEQ ID NO: 10continued) and the ORF sequence (SEQ ID NO: 6 continued) of LPLAT6 fromM. alpina strain 1S-4.

FIG. 6 is a graph showing the composition ratio of polyunsaturated fattyacids in cells when the expression of LPLAT6 or Δ5 fatty acid desaturaseis suppressed in M. alpina. In FIG. 6, the abbreviations have thefollowing meanings: GLA, γ-linolenic acid; DGLA, dihomo-γ-linolenicacid; ARA, arachidonic acid.

FIG. 7 is a graph showing the composition ratio of polyunsaturated fattyacids in triacylglycerol fractions when the expression of LPLAT6 or Δ5fatty acid desaturase is suppressed in M. alpina. In FIG. 7, theabbreviations have the following meanings: GLA, γ-linolenic acid; DGLA,dihomo-γ-linolenic acid; ARA, arachidonic acid.

FIG. 8 is a graph showing the composition ratio of polyunsaturated fattyacids in phospholipid fractions when the expression of LPLAT6 or Δ5fatty acid desaturase is suppressed in M. alpina. In FIG. 8, theabbreviations have the following meanings: GLA, γ-linolenic acid; DGLA,dihomo-γ-linolenic acid; ARA, arachidonic acid.

DESCRIPTION OF EMBODIMENT

The present invention relates to novel lysophospholipid acyltransferases(“LPLATs”) from the genus Mortierella characterized by transferring anacyl group between acyl-CoA and phospholipids in the biosyntheticprocess of arachidonic acid. The proteins of the present invention canact on lysophospholipids. The acyl donor is typically acyl-CoA, but notlimited thereto.

Embodiments of the present invention are specifically described below.

Nucleic Acids Encoding Lysophospholipid Acyltransferases of the PresentInvention

Lysophospholipid acyltransferases (LPLATs) encoded by the nucleic acidsof the present invention include LPLAT5 and 6 as typical examples.Unlike fatty acid compositions produced by hosts expressing known LPAATsfrom M. alpina, LPLAT5 and 6 could produce fatty acid compositionscharacterized by a high proportion of arachidonic acid, as explained inthe Examples below. Therefore, the LPLATs of the present inventionpreferably produce arachidonic acid with very high efficiency ascompared with known LPAATs from M. alpina.

Relationship of the cDNA, CDS, ORF of the nucleic acids encoding LPLAT5and LPLAT6 of the present invention and amino acid sequences issummarized in Table 1 below.

TABLE 1 LPLAT5 LPLAT6 Correspond- Correspond- ing region ing region inSEQ in SEQ SEQ ID NO: ID NO: 4 SEQ ID NO: ID NO: 9 ORF SEQ ID NO: 1161-1690 SEQ ID NO: 6 38-1756 Amino SEQ ID NO: 2 ***** SEQ ID NO: 7***** acid sequence CDS SEQ ID NO: 3 161-1693 SEQ ID NO: 8 38-1759 cDNASEQ ID NO: 4 ***** SEQ ID NO: 9 *****

In summary, sequences related to LPLAT5 of the present invention includeSEQ ID NO: 1 representing the sequence of the ORF region of LPLAT5; SEQID NO: 2 representing the amino acid sequence of LPLAT5; SEQ ID NO: 3representing the sequence of the CDS region of LPLAT5; SEQ ID NO: 4representing the nucleotide sequence of the cDNA; and SEQ ID NO: 5representing the genomic sequence. More specifically, nucleotides161-1693 of SEQ ID NO: 4 representing the cDNA sequence of LPLAT5corresponds to the CDS (SEQ ID NO: 3), and nucleotides 161-1690corresponds to the ORF (SEQ ID NO: 1). The cDNA sequence of LPLAT5 andits deduced amino acid sequence are shown in FIG. 2. The genomicsequence of (SEQ ID NO: 5) LPLAT5 contains two introns and exon regionscorresponding to nucleotides 1-314, 461-587 and 668-1759 of SEQ ID NO:5.

Similarly, sequences related to LPLAT6 of the present invention includeSEQ ID NO: 6 representing the sequence of the ORF region of LPLAT6; SEQID NO: 7 representing the amino acid sequence of LPLAT6; SEQ ID NO: 8representing the sequence of the CDS region of LPLAT6; SEQ ID NO: 9representing the nucleotide sequence of the cDNA; and SEQ ID NO: 10representing the genomic sequence. More specifically, nucleotides38-1759 of SEQ ID NO: 9 representing the cDNA sequence of LPLAT6corresponds to the CDS (SEQ ID NO: 8), and nucleotides 38-1756corresponds to the ORF (SEQ ID NO: 6). The cDNA sequence of LPLAT6 andits deduced amino acid sequence are shown in FIG. 3. The genomicsequence (SEQ ID NO: 10) of LPLAT6 contains one intron and exon regionscorresponding to nucleotides 1-1095 and 1318-1944 of SEQ ID NO: 10.

The nucleic acids of the present invention include single-stranded anddouble-stranded DNAs as well as RNA complements thereof, and may beeither naturally occurring or artificially prepared. DNAs include, butare not limited to, genomic DNAs, cDNAs corresponding to the genomicDNAs, chemically synthesized DNAs, PCR-amplified DNAs and combinationsthereof, as well as DNA/RNA hybrids, for example.

Preferred embodiments of the nucleic acids of the present inventioninclude (a) a nucleic acid that comprises the nucleotide sequence shownin SEQ ID NO: 1 or 6; (b) a nucleic acid that comprises a nucleotidesequence encoding a protein consisting of the amino acid sequence shownin SEQ ID NO: 2 or 7; (c) a nucleic acid that comprises the nucleotidesequence shown in SEQ ID NO: 4 or 9; or (d) a nucleic acid thatcomprises the nucleotide sequence shown in SEQ ID NO: 5 or 10, etc.

To obtain the above nucleotide sequences, nucleotide sequence data ofEST or genomic DNA from an organism having LPLAT activity can also besearched for nucleotide sequences encoding proteins sharing highidentity to a known protein having LPLAT activity. The organism havingLPLAT activity is preferably a lipid-producing fungus such as, but notlimited to, M. alpina.

To perform EST analysis, a cDNA library is first constructed. Proceduresfor cDNA library construction can be found in “Molecular Cloning, ALaboratory Manual 3rd ed.” (Cold Spring Harbor Press (2001)).Commercially available cDNA library construction kits may also be used.A procedure for constructing a cDNA library suitable for the presentinvention is as follows, for example. That is, an appropriate strain ofthe lipid-producing fungus M. alpina is inoculated into an appropriatemedium and precultured for an appropriate period. The cultures arecollected at appropriate time points during the main cultivation andcells are harvested to prepare total RNA. Total RNA can be preparedusing a known technique such as the guanidine hydrochloride/CsCl method.Poly(A)⁺RNA can be purified from the resulting total RNA using acommercially available kit. Further, a cDNA library can be constructedusing a commercially available kit. Then, ESTs can be obtained bydetermining the nucleotide sequences of any clones from the constructedcDNA library, by using primers designed to allow sequencing of an inserton a vector. For example, directional cloning can be performed when thecDNA library has been constructed using a ZAP-cDNA GigapackIII GoldCloning Kit (STRATAGENE).

As a result of homology analysis of SEQ ID NOs: 1 and 6 using BLASTXagainst amino acid sequences deposited in GenBank, the amino acidsequence deduced from SEQ ID NO: 1 shows homology to LPLAT homologs fromfungi and the amino acid sequence deduced from SEQ ID NO: 6 showshomology to LPLAT homologs from animals. The nucleotide sequenceidentity and amino acid sequence identity of the sequence showing thehighest identity to the ORF of each sequence were determined byclustalW, revealing that a lysophospholipid acyltransferase homolog fromSchizosaccharomyces pombe (GI:161085648) showed the lowest E-value orthe highest identity to SEQ ID NO: 1 and the nucleotide sequenceidentity and amino acid sequence identity in ORF were 43.2% and 33.3%,respectively. Similarly, a putative protein from Xenopus laevis(GI:56788919) showed the highest identity to SEQ ID NO: 6 and thenucleotide sequence identity and amino acid sequence identity in ORFwere 41.2% and 28.6%, respectively.

The nucleotide sequence identity and amino acid sequence identity in ORFbetween LPLAT5 and LPLAT6 are 40.0% and 19.1%, respectively.

The present invention also encompasses nucleic acids functionallyequivalent to nucleic acids that comprise the nucleotide sequences shownin SEQ ID NOs: 1 and 6 above (herein sometimes referred to as“nucleotide sequences of the present invention”) and nucleotidesequences encoding proteins consisting of the amino acid sequences shownin SEQ ID NO: 2 and 7 (herein sometimes referred to as “amino acidsequences of the present invention”). The expression “functionallyequivalent” means that a protein encoded by a nucleotide sequence of thepresent invention and a protein consisting of an amino acid sequence ofthe present invention have “lysophospholipid acyltransferase activity(LPLAT activity)”, “the activity of increasing the proportion ofarachidonic acid in the compositional ratio of fatty acids in a hosttransformed with a recombinant vector containing a nucleic acid encodinga protein of the present invention as compared with the proportion inthe compositional ratio of fatty acids in a host that has not beentransformed with the vector” (hereinafter sometimes referred to as “theactivity of increasing the proportion of arachidonic acid”)”, and/or“the activity involved in one or more conversions selected from thegroup consisting of the conversion from 18:1-CoA to 18:1-PL, conversionfrom 18:3(n-6)-PL to 18:3(n-6)-CoA, and conversion from DGLA-CoA toDGLA-PL (hereinafter sometimes referred to as “the activity involved inthe biosynthetic pathway of arachidonic acid”)”. Preferably, it meansthat the proteins have an activity similar to that of LPLAT5 and/or 6.

The “lysophospholipid acyltransferase (LPLAT) activity” of the presentinvention refers to the activity of transferring an acyl group betweenacyl-CoA and a lysophospholipid. “Lysophospholipid” refers to a lipidhaving one acyl group removed from a phospholipid. As used herein,lysophospholipids include, but not specifically limited to,lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC),lysophosphatidylserine (LPS), lysophosphatidylethanolamine (LPE),lysophosphatidylinositol (LPI), etc.

The LPLATs of the present invention may specifically act on onelysophospholipid or multiple specific lysophospholipids.

The LPLAT activity of the present invention can be assayed by knownmethods including, for example, the method described in J.B.C., 282(47),34288-34298 (2007).

The “activity of increasing the proportion of arachidonic acid” of thepresent invention refers to the activity of increasing the proportion ofarachidonic acid in the compositional ratio of fatty acids in a hosttransformed with a recombinant vector containing a nucleic acid of thepresent invention as compared with the proportion in the compositionalratio of fatty acids in a host that has not been transformed with thevector, as described above. Specifically, it refers to the activity ofincreasing the proportion of arachidonic acid in the compositional ratioof fatty acids of a host transformed with a recombinant vectorcontaining a nucleic acid that comprises a nucleotide sequence of thepresent invention or a nucleic acid that comprises a nucleotide sequenceencoding a protein consisting of an amino acid sequence of the presentinvention as compared with the proportion in the compositional ratio offatty acids in a host that has not been transformed with the vector. Theactivity can be assayed by known methods comprising, for example,transforming an expression vector pYE22m containing a nucleotidesequence of the present invention or the like into a recombinant host ofyeast Saccharomyces cerevisiae capable of producing arachidonic acid byintroducing and expressing Δ12 fatty acid desaturase gene, Δ6 fatty aciddesaturase gene, GLELO fatty acid elongase gene, and Δ5 fatty aciddesaturase gene; culturing the resulting transformant; harvesting thecultured cells; and subjecting them to fatty acid analysis by theprocedure described in the Examples below.

The “activity involved in the biosynthetic pathway of arachidonic acid”of the present invention refers to the activity involved in theconversion from 18:1-CoA to 18:1-PL, conversion from 18:3(n-6)-PL to18:3(n-6)-CoA, and/or conversion from DGLA-CoA to DGLA-PL. Preferably,the activity refers to the activity involved in the conversion from18:3(n-6)-PL to 18:3(n-6)-CoA, and/or the conversion from DGLA-CoA fromDGLA-PL. Here, 18:1-represents an oleoyl group, 18:3(n-6)-represents aγ-lilnoleyl group, DGLA-represents a dihomo-γ-lilnoleyl group, PLrepresents a phospholipid, and CoA represents coenzyme A, respectively.Therefore, DGLA-CoA refers to acyl-CoA containing a dihomo-γ-lilnoleylgroup, and DGLA-PL refers to a phospholipid containing adihomo-γ-lilnoleyl group, for example. The activity involved in thebiosynthetic pathway of arachidonic acid can be identified by observingthe conversion from each starting substrate to the produced substrate.Alternatively, it can be identified by observing that a protein of thepresent invention is overexpressed in a host or cell transformed with arecombinant vector containing a nucleic acid encoding a protein of thepresent invention or the expression of the protein is suppressed in acell capable of producing arachidonic acid. For example, it can beidentified by analyzing the compositional ratio of fatty acids in a hostor cell overexpressing a protein of the present invention or a host orcell underexpressing a protein of the present invention and observingchanges in the compositional ratio of fatty acids to assess theconversion from each starting substrate to the produced substrate by theprocedure described in the Examples below.

More preferably, the nucleotide sequences of the present invention orthe like are nucleic acids that comprise a nucleotide sequence encodinga protein having LPLAT activity, the activity of increasing theproportion of arachidonic acid, and/or the activity involved in thebiosynthetic pathway of arachidonic acid.

Still more preferably, the lysophospholipid acyltransferases (LPLATs)encoded by the nucleic acids of the present invention refer to enzymesbelonging to the membrane-bound O-acyltransferase (MBOAT) family amongLPLATs.

The “MBOAT family” refers to a family belonging to the protein of PFAMaccession number PF03062, and refers to a group of enzymes having atransmembrane domain in the amino acid sequence of glycerophospholipidacyltransferases. PFAM (pfam.sanger.ac.uk/) refers to a database ofprofiles obtained by protein family alignments provided by SangerInstitute. Each profile is composed of similar sequences and analyzed bya hidden Markov model. The protein family to which a desired proteinbelongs can be searched using keywords, the nucleic acid sequenceencoding the protein, the amino acid sequence of the protein, theaccession number and the like, in addition to the protein name ofinterest. Search using the nucleic acid sequences encoding the LPLATsobtained by the present invention or the amino acid sequences of theLPLATs reveals that the proteins belong to the MBOAT family of accessionnumber PF03062. Moreover, enzymes belonging to the MBOAT family have aconserved histidine residue in common at the active center, such as thehistidine residue at position 317 in the amino acid sequence of LPLAT5,and the histidine residue at position 456 in the amino acid sequence ofLPLAT6, for example.

Unlike the LPAAT family, the MBOAT family does not contain the LPAATmotif. The LPAAT motif refers to the conserved motif “HXXXXD (HX₄D)”occurring at four sites in the amino acid sequences of the LPAATproteins described in patent document 3. For example, the LPAAT motifoccurs at amino acid residues 115-120 of SEQ ID NO: 2 in patent document3 in LPAAT3 and at amino acid residues 115-120 of SEQ ID NO: 4 inLPAAT4, which are from the lipid-producing fungus M. alpina described inpatent document 3. However, the LPLAT proteins of the present inventioncontain no such motif.

In M. alpina, four LPLATs have been hitherto found (patent documents1-3), but no LPLAT enzyme belonging to the MBOAT family has been found.Thus, the LPLATs of the present invention are most preferably LPLATsbelonging to the MBOAT family and having the above activity of thepresent invention.

Nucleic acids functionally equivalent to the nucleic acids of thepresent invention as described above include a nucleic acid thatcomprises the nucleotide sequence of any one of (a)-(e) below. As usedin reference to the nucleotide sequences herein below, “the aboveactivity of the present invention” refers to the “LPLAT activity, theactivity of increasing the proportion of arachidonic acid, and/or theactivity involved in the biosynthetic pathway of arachidonic acid”defined above.

(a) A nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence with deletion, substitutionor addition of one or more amino acids in the amino acid sequence shownin SEQ ID NO: 2 or 7, and having the above activity of the presentinvention.

The nucleic acid of the present invention comprises a nucleotidesequence encoding a protein consisting of an amino acid sequence withdeletion, substitution or addition of one or more amino acids in theamino acid sequence shown in SEQ ID NO: 2 or 7, and having the aboveactivity of the present invention. The “above activity of the presentinvention” is as described above.

Specifically, it comprises a nucleotide sequence encoding a proteinconsisting of an amino acid sequence with deletion, substitution and/oraddition of one or more (preferably one or several (e.g., 1-400, 1-200,1-100, 1-50, 1-30, 1-25, 1-20, 1-15, more preferably 10, 9, 8, 7, 6, 5,4, 3, 2, or 1)) amino acids in the amino acid sequence shown in SEQ IDNO: 2 or 7; and having the above activity of the present invention.Here, the expression “amino acid sequence with deletion, substitution,and/or addition” means that one or more amino acids are deleted,substituted and/or added at one or more random positions in the sameamino acid sequence. Two or more of the deletion, substitution and/oraddition may occur at the same time, but the number of the deletion,substitution and/or addition is preferably smaller, in general.

In the above modifications, the substitution is preferably conservative.Conservative substitution refers to replacement of a particular aminoacid residue by another residue having similar physicochemicalcharacteristics, and may be any substitution that does not substantiallyaffect the structural characteristics of the original sequence, e.g., itmay be any substitution so far as the substituted amino acids do notdisrupt a helix present in the original sequence or do not disrupt anyother type of secondary structure characteristic of the originalsequence.

Conservative substitution is typically introduced by synthesis inbiological systems or chemical peptide synthesis, preferably by chemicalpeptide synthesis. Substituents here may include unnatural amino acidresidues, as well as peptidomimetics, and reversed or inverted forms ofamino acid sequences in which unsubstituted regions are reversed orinverted.

A non-limitative list of groups of amino acid residues that can besubstituted for each other is shown below.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine,t-butylalanine and cyclohexylalanine;Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid and 2-aminosuberic acid;Group C: asparagine and glutamine;Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid and2,3-diaminopropionic acid;Group E: proline, 3-hydroxyproline and 4-hydroxyproline;Group F: serine, threonine and homoserine; andGroup G: phenylalanine and tyrosine.

Non-conservative substitution may include replacement of a member of oneof the above groups by a member of another group, in which case thehydropathic indices of amino acids (amino acid hydropathic indices)should preferably be considered in order to retain biological functionsof the proteins of the present invention (Kyte et al., J. Mol. Biol.,157:105-131(1982)).

Non-conservative substitution may also include amino acid replacementbased on hydrophilicity.

In the specification and drawings herein, nucleotide and amino acidnotions and abbreviations are based on the IUPAC-IUB Commission onBiochemical Nomenclature or protocols conventionally used in the art asdescribed, for example, in Immunology-A Synthesis (second edition,edited by E. S. Golub and D. R. Gren, Sinauer Associates, Sunderland,Mass. (1991)). Any optical isomers of amino acids that may exist referto L-isomers, unless otherwise specified.

Stereoisomers of the above amino acids such as D-amino acids, unnaturalamino acids such as α,α-disubstituted amino acids, N-alkylamino acids,lactic acid, and other non-canonical amino acids may also be componentsof the proteins of the present invention.

Proteins are herein written with the amino-terminus on the left and thecarboxy-terminus on the right in accordance with standard usage andconvention in the art. Similarly, single-stranded polynucleotidesequences are written with the 5′-end on the left end, anddouble-stranded polynucleotide sequences are written with the 5′-end ofone strand on the left in general, unless otherwise specified.

One skilled in the art will be able to design and generate suitablevariants of the proteins described herein using techniques known in theart. For example, one may identify suitable areas of the proteinmolecule that may be structurally changed without destroying biologicalactivity of a protein of the present invention by targeting areas notbelieved to be important for the biological activity of the protein ofthe present invention. Also, one may identify residues and areasconserved between similar proteins. Furthermore, one will be able tointroduce conservative amino acid substitutions into areas that may beimportant for the biological activity or structure of the protein of thepresent invention without destroying the biological activity and withoutadversely affecting the polypeptide structure of the protein.

One skilled in the art can perform so-called structure-function studiesidentifying residues in a peptide similar to a peptide of a protein ofthe present invention that are important for biological activity orstructure of the protein of the present invention, and comparing theamino acid residues in the two peptides to predict which residues in aprotein similar to the protein of the present invention are amino acidresidues that correspond to amino acid residues that are important forbiological activity or structure. Further, one may choose variants thatretain the biological activity of the protein of the present inventionby opting for chemically similar amino acid substitutions for suchpredicted amino acid residues. One skilled in the art can also analyzethe three-dimensional structure and amino acid sequence of the variantsof the protein. In view of the analytical results, one may furtherpredict the alignment of amino acid residues with respect to thethree-dimensional structure of the protein. Based on the analyticalresults as described above, one skilled in the art may also generatevariants containing no changes to amino acid residues predicted to be onthe surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate variants containing a single amino acidsubstitution among the amino acid residues constituting the protein ofthe present invention. The variants can be screened by known assays togather information about the individual variants. As a result, one mayevaluate usefulness of the individual amino acid residues constitutingthe protein of the present invention by comparing variants containing achange to a particular amino acid residue to assess whether they showreduced biological activity as compared with the biological activity ofthe protein of the present invention, or they show no such biologicalactivity, or they show unsuitable activity inhibiting the biologicalactivity of the protein of the present invention. Moreover, based oninformation gathered from such routine experiments, one skilled in theart can readily analyze undesirable amino acid substitutions forvariants of the protein of the present invention either alone or incombination with other mutations.

As described above, proteins consisting of an amino acid sequence withdeletion, substitution or addition of one or more amino acids in theamino acid sequence shown in SEQ ID NO: 2 or 7 can be prepared by suchtechniques as site-directed mutagenesis as described in “MolecularCloning, A Laboratory Manual 3rd ed.” (Cold Spring Harbor Press (2001));“Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997);Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-92; Kunkel (1988)Method. Enzymol. 85: 2763-6, etc. Preparation of such variantscontaining amino acid deletions, substitutions or additions or the likecan be carried out by known procedures such as e.g., the Kunkel methodor the Gapped duplex method, using a mutation-introducing kit based onsite-directed mutagenesis such as e.g., a QuikChange™ Site-DirectedMutagenesis Kit (Stratagene), a GeneTailor™ Site-Directed MutagenesisSystem (Invitrogen) or a TaKaRa Site-Directed Mutagenesis System(Mutan-K, Mutan-Super Express Km, etc.; Takara Bio Inc.).

In addition to the site-directed mutagenesis mentioned above, techniquesfor introducing deletion, substitution or addition of one or more aminoacids in the amino acid sequences of proteins while retaining theiractivity include treatment of a gene with a mutagen, and selectivecleavage of a gene to remove, substitute or add a selected nucleotidefollowed by ligation.

A nucleic acid of the present invention preferably comprises anucleotide sequence encoding a protein consisting of an amino acidsequence with deletion, substitution or addition of 1-50 amino acids inthe amino acid sequence shown in SEQ ID NO: 2 or 7, and having the aboveactivity of the present invention. There is no limitation on the numberor sites of amino acid changes or modifications in the proteins of thepresent invention so far as the above activity of the present inventionis retained. The method for assaying the above activity of the presentinvention is as described above.

(b) A nucleic acid that hybridizes under stringent conditions to anucleic acid consisting of a nucleotide sequence complementary to thenucleotide sequence consisting of SEQ ID NO: 1 or 6 and that comprises anucleotide sequence encoding a protein having the above activity of thepresent invention.

The nucleic acid of the present invention hybridizes under stringentconditions to a nucleic acid consisting of a nucleotide sequencecomplementary to the nucleotide sequence consisting of SEQ ID NO: 1 or 6and comprises a nucleotide sequence encoding a protein having the aboveactivity of the present invention. The “above activity of the presentinvention” is as described above.

The above nucleotide sequence can be obtained from a cDNA library and agenomic library or the like by a known hybridization technique such ascolony hybridization, plaque hybridization or Southern blotting using aprobe prepared from an appropriate fragment by a method known to thoseskilled in the art.

Detailed procedures for hybridization can be found in “MolecularCloning, A Laboratory Manual 3rd ed.” (Cold Spring Harbor Press (2001);especially Sections 6-7); “Current Protocols in Molecular Biology” (JohnWiley & Sons (1987-1997); especially Sections 6.3-6.4); “DNA Cloning 1:Core Techniques, A Practical Approach 2nd ed.” (Oxford University(1995); especially Section 2.10 for hybridization conditions), etc.

The strength of hybridization conditions is determined primarily byhybridization conditions, more preferably by hybridization conditionsand washing conditions. As used herein, “stringent conditions” includemoderately or highly stringent conditions.

Specifically, moderately stringent conditions include, for example,hybridization conditions of 1×SSC-6×SSC at 42° C.-55° C., morepreferably 1×SSC-3×SSC at 45° C.-50° C., most preferably 2×SSC at 50° C.When the hybridization solution contains about 50% formamide, forexample, temperatures 5-15° C. below the temperatures indicated aboveare used. Washing conditions include 0.5×SSC-6×SSC at 40° C.-60° C.During hybridization and washing, typically 0.05%-0.2%, preferably about0.1% SDS may be added.

Highly stringent (high stringent) conditions include hybridizationand/or washing at higher temperatures and/or lower salt concentrationsthan those of the moderately stringent conditions. For example,hybridization conditions include 0.1×SSC-2×SSC at 55° C.-65° C., morepreferably 0.1×SSC-1×SSC at 60° C.-65° C., most preferably 0.2×SSC at63° C. Washing conditions include 0.2×SSC-2×SSC at 50° C.-68° C., morepreferably 0.2×SSC at 60-65° C.

Hybridization conditions specifically used in the present inventioninclude for example, but are not limited to, prehybridization in 5×SSC,1% SDS, 50 mM Tris-HCl (pH 7.5) and 50% formamide at 42° C. followed byhybridization with a probe at 42° C. overnight, and then washing threetimes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes.

Commercially available hybridization kits using no radioactive probe canalso be used. Specifically, hybridization may be performed using a DIGnucleic acid detection kit (Roche Diagnostics) or an ECL direct labeling& detection system (Amersham), etc.

A nucleic acid included in the present invention preferably hybridizesunder conditions of 2×SSC, 50° C. to a nucleic acid consisting of anucleotide sequence complementary to the nucleotide sequence consistingof SEQ ID NO: 1 or 6 and comprises a nucleotide sequence encoding aprotein having the above activity of the present invention.

(c) A nucleic acid that comprises a nucleotide sequence sharing anidentity of 80% or more with the nucleotide sequence consisting of SEQID NO: 1 or 6 and encoding a protein having the above activity of thepresent invention. The nucleic acid of the present invention comprises anucleotide sequence having at least 80% identity to the nucleotidesequence shown in SEQ ID NO: 1 or 6 and encoding a protein having theabove activity of the present invention. The “above activity of thepresent invention” is as described above.

Preferably, the nucleic acid comprises a nucleotide sequence having atleast 80%, more preferably 85%, still more preferably 90% (e.g., 92% ormore, still more preferably 95% or more, even 97%, 98% or 99%) identityto the nucleotide sequence shown in SEQ ID NO: 1 or 6 and encoding aprotein having the above activity of the present invention.

The percent identity between two nucleic acid sequences can bedetermined by visual inspection and mathematical calculation, orpreferably by comparing sequence information of the two nucleic acidsusing a computer program. Computer programs for sequence comparisoninclude, for example, the BLASTN program (Altschul et al. (1990) J. Mol.Biol. 215: 403-10) version 2.2.7 available from the website of the U.S.National Library of Medicine:www.ncbi.nlm.nih.gov/blast/b12seq/bls.html, or the WU-BLAST 2.0algorithm, etc. Standard default parameter settings for WU-BLAST 2.0 areavailable at the following Internet site: blast.wustl.edu.

(d) A nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence sharing an identity of 80%or more with the amino acid sequence consisting of SEQ ID NO: 2 or 7 andhaving the above activity of the present invention.

The nucleic acid of the present invention comprises a nucleotidesequence encoding a protein consisting of an amino acid sequence sharingan identity of 80% or more with the amino acid sequence consisting ofSEQ ID NO: 2 or 7 and having the above activity of the presentinvention. The “above activity of the present invention” is as describedabove.

Specifically, the amino acid sequence has 80% or more, preferably 85% ormore, more preferably 90%, still more preferably 95% or more, even morepreferably 97% (e.g., 98%, even 99%) or more identity to the amino acidsequence of SEQ ID NO: 2 or 7 or the like.

The nucleic acid of the present invention preferably comprises anucleotide sequence encoding a protein consisting of an amino acidsequence sharing an identity of 95% or more with the amino acid sequenceconsisting of SEQ ID NO: 2 or 7 and having the above activity of thepresent invention. More preferably, the nucleic acid comprises anucleotide sequence encoding a protein consisting of an amino acidsequence sharing an identity of 98% or more with the amino acid sequenceconsisting of SEQ ID NO: 2 or 7 and having the above activity of thepresent invention.

The percent identity between two amino acid sequences can be determinedby visual inspection and mathematical calculation. Alternatively, thepercent identity can be determined by using a computer program. Suchcomputer programs include, for example, BLAST, FASTA (Altschul et al.,J. Mol. Biol., 215:403-410 (1990)) and ClustalW, etc. In particular,various conditions (parameters) for an identity search with the BLASTprogram are described by Altschul et al. (Nucl. Acids. Res., 25, p.3389-34.02, 1997) and publicly available from the website of theNational Center for Biotechnology Information (NCBI) or the DNA DataBank of Japan (DDBJ) (BLAST Manual, Altschul et al., NCB/NLM/NIHBethesda, Md. 20894; Altschul et al.). The percent identity can also bedetermined using genetic information processing programs such as GENETYXVer.7 (Genetyx), DNASIS Pro (Hitachisoft), Vector NTI (Infomax), etc.

Certain alignment schemes for aligning amino acid sequences may resultin the matching of even a specific short region of the sequences, andthereby it is possible to detect a region with very high sequenceidentity in such a small aligned region, even when there is nosignificant relationship between the full-length sequences used. Inaddition, the BLAST algorithm may use the BLOSUM62 amino acid scoringmatrix and optional parameters as follows: (A) inclusion of a filter tomask off segments of the query sequence that have low compositionalcomplexity (as determined by the SEG program of Wootton and Federhen(Computers and Chemistry, 1993); also see Wootton and Federhen, 1996,“Analysis of compositionally biased regions in sequence databases,”Methods Enzymol., 266: 554-71) or segments consisting ofshort-periodicity internal repeats (as determined by the XNU program ofClaverie and States (Computers and Chemistry, 1993)), and (B) astatistical significance threshold for reporting matches againstdatabase sequences, or E-score (the expected probability of matchesbeing found merely by chance according to the stochastic model of Karlinand Altschul, 1990; if the statistical significance ascribed to a matchis greater than this E-score threshold, the match will not be reported).

(e) A nucleic acid that hybridizes under stringent conditions to anucleic acid consisting of a nucleotide sequence complementary to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 or 7 and that comprises a nucleotidesequence encoding a protein having the above activity of the presentinvention.

The nucleic acid of the present invention hybridizes under stringentconditions to a nucleic acid consisting of a nucleotide sequencecomplementary to a nucleotide sequence encoding a protein consisting ofthe amino acid sequence shown in SEQ ID NO: 2 or 7 and comprises anucleotide sequence encoding a protein having the above activity of thepresent invention.

The “above activity of the present invention” and hybridizationconditions are as described above.

Further, the nucleic acids of the present invention also include anucleic acid that comprises a nucleotide sequence with deletion,substitution or addition of one or more nucleotides in the nucleotidesequence consisting of SEQ ID NO: 1 or 6, and encoding a protein havingthe above activity of the present invention. Specifically, it is alsopossible to use a nucleic acid which comprises a nucleotide sequencewith deletion, substitution or addition of one or more (preferably oneor several (e.g., 1-1500, 1-1000, 1-500, 1-300, 1-250, 1-200, 1-150,1-100, 1-50, 1-30, 1-25, 1-20, 1-15, more preferably 10, 9, 8, 7, 6, 5,4, 3, 2, or 1)) nucleotides in the nucleotide sequence shown in SEQ IDNO:1 or 6, and encoding a protein having the above activity of thepresent invention. As used here, the expression “nucleotide sequencewith deletion, substitution or addition” means that one or morenucleotides are deleted, substituted and/or added at one or more randompositions in the same nucleotide sequence. Two or more of the deletion,substitution and/or addition may occur at the same time, but the numberof the deletion, substitution and/or addition is preferably smaller, ingeneral.

Preferred embodiments of the nucleic acids of the present invention alsoinclude a nucleic acid of any one of (a)-(d) below:

(a) a nucleic acid that comprises the nucleotide sequence shown in SEQID NO: 1 or 6 or a partial sequence thereof;(b) a nucleic acid that comprises a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO: 2 or 7 or a partial sequencethereof;(c) a nucleic acid that comprises the nucleotide sequence shown in SEQID NO: 4 or 9 or a partial sequence thereof;(d) a nucleic acid that comprises the nucleotide sequence shown in SEQID NO: 5 or 10 or a partial sequence thereof.

The nucleic acids defined as (a) a nucleic acid that comprises thenucleotide sequence shown in SEQ ID NO: 1 or 6; (b) a nucleic acid thatcomprises a nucleotide sequence encoding a protein consisting of theamino acid sequence shown in SEQ ID NO: 2 or 7; and (c) a nucleic acidthat comprises the nucleotide sequence shown in SEQ ID NO: 4 or 9 are asdescribed above. The partial sequence of the above sequences are regionscontained in the above nucleotide sequences including ORFs, CDSs,biologically active regions, regions used as primers as described below,and regions capable of serving as probes, and may be naturally occurringor artificially prepared.

The nucleic acids of the present invention are preferably nucleic acidsencoding a protein belonging to the membrane-bound O-acyltransferasefamily. The “membrane-bound O-acyltransferase family” is as describedabove.

The nucleic acids of the present invention also include:

(1) a nucleic acid of any one of (a)-(e) below:(a) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence with deletion, substitutionor addition of one or more amino acids in the amino acid sequence shownin SEQ ID NO: 2 or 7;(b) a nucleic acid that hybridizes under stringent conditions to anucleic acid consisting of a nucleotide sequence complementary to thenucleotide sequence consisting of SEQ ID NO: 1 or 6;(c) a nucleic acid that comprises a nucleotide sequence sharing anidentity of 80% or more with the nucleotide sequence consisting of SEQID NO: 1 or 6;(d) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence sharing an identity of 80%or more with the amino acid sequence consisting of SEQ ID NO: 2 or 7;(e) a nucleic acid that hybridizes under stringent conditions to anucleic acid consisting of a nucleotide sequence complementary to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 or 7; and(2) the nucleic acid of (1), which is any one of (a)-(e) below:(a) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence with deletion, substitutionor addition of 1-50 amino acids in the amino acid sequence shown in SEQID NO: 2 or 7;(b) a nucleic acid that hybridizes under conditions of 2×SSC, 50° C. toa nucleic acid consisting of a nucleotide sequence complementary to thenucleotide sequence consisting of SEQ ID NO: 1 or 6;(c) a nucleic acid that comprises a nucleotide sequence sharing anidentity of 90% or more with the nucleotide sequence consisting of SEQID NO: 1 or 6;(d) a nucleic acid that comprises a nucleotide sequence encoding aprotein consisting of an amino acid sequence sharing an identity of 90%or more with the amino acid sequence consisting of SEQ ID NO: 2 or 7;(e) a nucleic acid that hybridizes under conditions of 2×SSC, 50° C. toa nucleic acid consisting of a nucleotide sequence complementary to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 or 7.

Lysophospholipid Acyltransferase Proteins of the Present Invention

The proteins of the present invention are characterized in that theyhave “lysophospholipid acyltransferase activity (LPLAT activity)”, “theactivity of increasing the proportion of arachidonic acid”, and/or “theactivity involved in the biosynthetic pathway of arachidonic acid”. Theproteins of the present invention may be naturally occurring orartificially prepared.

The proteins of the present invention are preferably LPLAT5 and LPLAT6consisting of the amino acid sequence shown in SEQ ID NO: 2 or 7.Further, the present invention also encompasses variants of LPLAT5 andLPLAT6, i.e. variants satisfying the criteria: having “lysophospholipidacyltransferase activity (LPLAT activity)”, “the activity of increasingthe proportion of arachidonic acid”, and/or “the activity involved inthe biosynthetic pathway of arachidonic acid”.

The “lysophospholipid acyltransferase activity”, “the activity ofincreasing the proportion of arachidonic acid” and “the activityinvolved in the biosynthetic pathway of arachidonic acid” are asdescribed above in the section “Nucleic acids encoding lysophospholipidacyltransferases of the present invention”. As used herein below, the“above activity of the present invention” refers to the “LPLAT activity,the activity of increasing the proportion of arachidonic acid, and/orthe activity involved in the biosynthetic pathway of arachidonic acid”defined above.

The proteins of the present invention include a protein of (a) or (b)below:

(a) a protein consisting of an amino acid sequence with deletion,substitution or addition of one or more amino acids in the amino acidsequence of SEQ ID NO: 2 or 7, and having the above activity of thepresent invention;(b) a protein consisting of an amino acid sequence sharing an identityof 80% or more with the amino acid sequence consisting of SEQ ID NO: 2or 7 and having the above activity of the present invention.

The definitions of “an amino acid sequence with deletion, substitutionor addition of one or more amino acids in an amino acid sequence” and“identity of 80% or more” are as explained above in the section “Nucleicacids encoding lysophospholipid acyltransferases of the presentinvention”.

The proteins of the present invention also include a variant of aprotein encoded by a nucleic acid comprising the nucleotide sequence ofSEQ ID NO: 1 or 6, or a protein of an amino acid sequence with deletion,substitution or addition of one or more amino acids in the amino acidsequence shown in SEQ ID NO: 2 or 7 or otherwise modified, or a modifiedprotein having a modified amino acid side chain, or a fusion proteinwith another protein and having the above activity of the presentinvention.

The proteins of the present invention may be artificially prepared bychemical synthesis techniques such as Fmoc method(fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonylmethod). They can also be chemically synthesized using a peptidesynthesizer available from Advanced ChemTech, Perkin Elmer, Pharmacia,Protein Technology Instrument, Synthecell-Vega, PerSeptive, ShimadzuCorporation or the like.

Moreover, the proteins of the present invention are preferably proteinsbelonging to the membrane-bound O-acyltransferase family. The definitionor the like of the “membrane-bound O-acyltransferase family” is asexplained above in the section “Nucleic acids encoding lysophospholipidacyltransferases of the present invention”.

The proteins of the present invention also include:

(1) a protein of (a) or (b) below:(a) a protein consisting of an amino acid sequence with deletion,substitution or addition of one or more amino acids in the amino acidsequence of SEQ ID NO: 2 or 7;(b) a protein consisting of an amino acid sequence sharing an identityof 80% or more with the amino acid sequence consisting of SEQ ID NO: 2or 7;(2) the protein of (1), which is (a) or (b) below:(a) a protein consisting of an amino acid sequence with deletion,substitution or addition of 1-50 amino acids in the amino acid sequenceof SEQ ID NO: 2 or 7;(b) a protein consisting of an amino acid sequence sharing an identityof 90% or more with the amino acid sequence consisting of SEQ ID NO: 2or 7.

Cloning of the Nucleic Acids of the Present Invention

The nucleic acids encoding the LPLAT proteins of the present inventioncan be cloned by, for example, screening from a cDNA library using anappropriate probe. They can also be cloned by PCR amplification withappropriate primers followed by ligation to an appropriate vector. Theresulting clone may further be subcloned into another vector.

For example, commercially available plasmid vectors can be used, such aspBlue-Script™ SK (+) (Stratagene), pGEM-T (Promega), pAmp (TM:Gibco-BRL), p-Direct (Clontech) and pCR2.1-TOPO (Invitrogen). Foramplification by PCR, any regions of the nucleotide sequences shown inSEQ ID NO: 1 or 6 and the like may be used as primers, including theprimers shown in Example 1 below, for example. PCR is performed byadding the above primers and a heat-resistant DNA polymerase or the liketo act on cDNA prepared from M. alpina cells. The above procedure can bereadily accomplished by those skilled in the art according to “MolecularCloning, A Laboratory Manual 3rd ed.” (Cold Spring Harbor Press (2001))or the like.

The resulting PCR product can be purified using known methods. Forexample, purification methods include those using kits such as GENECLEAN(Funakoshi Co., Ltd.), QIAquick PCR purification Kits (QIAGEN),ExoSAP-IT (GE Healthcare Bio-Sciences); or using DEAE-cellulose filtersor dialysis tubes, etc. When an agarose gel is used, DNA fragments aresubjected to agarose gel electrophoresis and the DNA fragments reexcised from the agarose gel, followed by purification with GENECLEAN(Funakoshi Co., Ltd.), QIAquick Gel extraction Kits (QIAGEN), afreeze-squeeze method, etc.

The nucleotide sequences of the cloned nucleic acids can be determinedusing a nucleotide sequencer.

Construction of Expression Vectors of the Present Invention andPreparation of Transformed Cells

The present invention also provides recombinant vectors containing anucleic acid encoding an LPLAT protein of the present invention. Thepresent invention further provides cells transformed with therecombinant vectors.

Such recombinant vectors and transformants can be obtained as follows.That is, a plasmid carrying a nucleic acid encoding an LPLAT protein ofthe present invention is digested with restriction endonucleases. Therestriction endonucleases used include for example, but not limited to,EcoRI, KpnI, BamHI and SalI, etc. The plasmid may be blunt-ended by T4polymerase treatment. The digested DNA fragment is purified by agarosegel electrophoresis. This DNA fragment may be inserted into anexpression vector by a known method, thereby giving a vector forexpressing the LPLAT protein. This expression vector is transformed intoa host to prepare a transformant, which is used for the expression of adesired protein.

The expression vector and host here are not specifically limited so faras a desired protein can be expressed, and suitable hosts include fungi,bacteria, plants and animals or cells thereof, for example. Fungiinclude filamentous fungi such as the lipid-producing fungus M. alpina,yeast such as S. cerevisiae (Saccharomyces cerevisiae), etc. Bacteriainclude Escherichia coli, Bacillus subtilis, etc. Further, plantsinclude oil-producing plants such as rapeseed, soybean, cottonseed,safflower and flax.

Lipid-producing fungi that can be used include, for example, the strainsdescribed in MYCOTAXON, Vol. XLIV, NO. 2, pp. 257-265 (1992),specifically microorganisms belonging to the genus Mortierella,including microorganisms belonging to the subgenus Mortierella such asMortierella elongata (M. elongata) W08570, Mortierella exigua (M.exigua) IFO8571, Mortierella hygrophila (M. hygrophila) IFO5941,Mortierella alpina IFO8568, ATCC16266, ATCC32221, ATCC42430, CBS 219.35,CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS528.72, CBS529.72,CBS608.70, CBS754.68, or microorganisms belonging to the subgenusMicromucor such as Mortierella isabellina (M. isabellina) CBS194.28,IFO6336, IFO7824, IFO7873, IFO7874, IFO8286, IFO8308, IFO7884,Mortierella nana (M. nana) IFO8190, Mortierella ramanniana (M.ramanniana) IFO5426, IFO8186, CBS112.08, CBS212.72, IFO7825, IFO8184,IFO8185, IFO8287, Mortierella vinacea (M. vinacea) CBS236.82. Amongothers, M. alpina is preferred.

When a fungus is used as a host, the vector preferably has a structurethat allows a nucleic acid of the present invention to self-replicate inthe host or to be inserted onto a chromosome of the fungus. Also, itpreferably contains a promoter and a terminator. When M. alpina is usedas a host, the expression vector may be, for example, pD4, pDuraSC,pDura5 or the like. Any promoter that can be expressed in the host maybe used, including M. alpina-derived promoters such as the promoter ofthe histone H4.1 gene, the promoter of the GAPDH(glyceraldehyde-3-phosphate dehydrogenase) gene and the promoter of theTEF (translation elongation factor) gene.

Techniques for transforming a recombinant vector into filamentous fungisuch as M. alpina include electroporation, the spheroplast method,particle delivery, and direct microinjection of DNA into nuclei, etc.When an auxotrophic host strain is used, transformed strains can beobtained by selecting strains growing on a selective medium lacking itsessential nutrients. When a drug resistance marker gene is used fortransformation, cell colonies showing drug resistance can be obtained byculturing in a selective medium containing the drug.

When yeast is used as a host, the expression vector may be, for example,pYE22m or the like. Commercially available yeast expression vectors suchas pYES (Invitrogen) and pESC (STRATAGENE) may also be used. Yeast hostssuitable for the present invention include, but are not limited to, S.cerevisiae strain EH13-15 (trp1, MATα), etc. Promoters used include, forexample, those derived from yeast or the like, such as GAPDH promoter,GAL1 promoter and GAL10 promoter.

Techniques for transforming a recombinant vector into yeast include, forexample, the lithium acetate method, electroporation, the spheroplastmethod, dextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion, encapsulation of(one or more) polynucleotide (s) in liposomes, and direct microinjectionof DNA into nuclei, etc.

When a bacterium such as E. coli is used as a host, the expressionvector may be, for example, pGEX, pUC18 or the like available fromPharmacia. Promoters that can be used include those derived from E.coli, phages and the like, such as trp promoter, lac promoter, PLpromoter and PR promoter, for example. Techniques for transforming arecombinant vector into bacteria include, for example, electroporationand the calcium chloride method.

Methods for Preparing Fatty Acid Compositions of the Present Invention

The present invention provides methods for preparing a fatty acidcomposition from the transformed cell described above, i.e., methods forpreparing a fatty acid composition from cultures of the transformedcell. Specifically, it can be prepared by the procedure described below.However, the present methods are not limited to the procedures below,but can also be carried out by using other conventional knownprocedures.

Any liquid medium (culture medium) may be used for culturing an organismexpressing a protein of the present invention so far as it hasappropriate pH and osmotic pressure and contains nutrients required forgrowth of each host, trace elements, and biological materials such assera or antibiotics. For example, culture media that can be used foryeast cells transformed to express LPLAT 5 and 6 include, but notlimited to, SC-Trp, Leu, Ura medium, YPD medium, YPD5 medium and thelike.

Any culture conditions suitable for host growth and for stablymaintaining the generated enzyme may be used, and specifically,individual conditions can be adjusted, including anaerobicity,incubation period, temperature, humidity, static or shaking culture,etc. Cultivation may be performed under the same conditions (one-stepculture) or may be so-called two-step or three-step culture using two ormore different culture conditions, but two-step culture and the like arepreferred for large-scale culture, because of high culture efficiency.

Fatty Acid Compositions of the Present Invention

The present invention also provides fatty acid compositions comprisingan assembly of one or more fatty acids in a cell expressing an LPLATprotein of the present invention, characterized in that the proportionof arachidonic acid in compositional ratio of fatty acids in the fattyacid composition is higher than the proportion of arachidonic acid infatty acid compositions obtained by culturing non-transformed hosts.Preferably, it provides fatty acid compositions obtained by culturing atransformed cell expressing LPLAT5 and 6 of the present invention. Inthe Examples below, the proportion of arachidonic acid in an-arachidonicacid-producing yeast transformed with LPLAT5 or 6 increased at least1.5-fold as compared with the proportion of arachidonic acid in thecontrol fatty acid composition.

The fatty acids may be free fatty acids or those composingtriglycerides, phospholipids or the like.

The fatty acids contained in the fatty acid compositions of the presentinvention are linear or branched monocarboxylic acids with long-chaincarbohydrates, including for example, but not limited to, myristic acid(tetradecanoic acid) (14:0), myristoleic acid (tetradecenoic acid)(14:1), palmitic acid (hexadecanoic acid) (16:0), palmitoleic acid(9-hexadecenoic acid) (16:1), stearic acid (octadecanoic acid) (18:0),oleic acid (cis-9-octadecenoic acid) (18:1 (9) or sometimes simplyreferred to as 18:1), vaccenic acid (11-octadecenoic acid) (18:1 (11)),linoleic acid (cis,cis-9,12 octadecadienoic acid) (18:2 (9,12) orsometimes simply referred to as 18:2), α-linolenic acid(9,12,15-octadecatrienoic acid) (18:3 (9,12,15)), γ-linolenic acid(6,9,12-octadecatrienoic acid) (18:3 (6,9,12), GLA or sometimes referredto as 18:3(n-6)), stearidonic acid (6,9,12,15-octadecatetraenoic acid)(18:4 (6,9,12,15)), arachidic acid (icosanoic acid) (20:0),(8,11-icosadienoic acid) (20:2 (8,11)), mead acid (5,8,11-icosatrienoicacid) (20:3 (5,8,11)), dihomo-γ-linolenic acid (8,11,14-icosatrienoicacid) (20:3 (8,11,14) or sometimes referred to as DGLA), arachidonicacid (5,8,11,14-icosatetraenoic acid) (20:4 (5,8,11,14) or sometimesreferred to as ALA), eicosatetraenoic acid (8,11,14,17-icosatetraenoicacid) (20:4 (8,11,14,17)), eicosapentaenoic acid(5,8,11,14,17-icosapentaenoic acid) (20:5 (5,8,11,14,17)), behenic acid(docosanoic acid) (22:0), (7,10,13,16-docosatetraenoic acid) (22:4(7,10,13,16)), (7,10,13,16,19-docosapentaenoic acid) (22:5(7,10,13,16,19)), (4,7,10,13,16-docosapentaenoic acid) (22:5(4,7,10,13,16)), (4,7,10,13,16,19-docosahexaenoic acid) (22:6(4,7,10,13,16,19)), lignoceric acid (tetradocosanoic acid) (24:0),nervonic acid (cis-15-tetracosenoic acid) (24:1), cerotic acid(hexadocosanoic acid) (26:0), etc. The chemical names shown above arecommon names defined by the IUPAC Biochemical Nomenclature, and eachfollowed by the systematic name and then the number of carbon atoms andthe number and positions of double bonds in parentheses.

The fatty acid composition of the present inventions may be composed ofany number and any type of fatty acids so far as they comprise acombination of one or more of the fatty acids listed above.

Lyophilized cells obtained by the methods for preparing fatty acidcompositions of the present invention described above are stirred with achloroform/methanol mixture prepared in a suitable ratio, and thenheated for a suitable period. Further, separation of the cells bycentrifugation and solvent recovery are repeated several times. Then,lipids are dried by a suitable method and then dissolved in a solventsuch as chloroform. An aliquot of this sample is collected and fattyacids in the cells are converted into methyl esters using methanolicHCl, then extracted with hexane, and hexane is distilled off and theresidue is analyzed by gas chromatography.

The proportion of arachidonic acid in the compositional ratio of fattyacids of the fatty acid composition obtained by culturing a celltransformed with a recombinant vector containing a nucleic acid of thepresent invention is higher than the proportion of arachidonic acid inknown LPLAT fatty acid compositions. This is attributed to the fact thatthe LPLATs of the present invention can increase the conversion of fattyacids requiring acyl transfer from acyl-CoA to phospholipids or fromphospholipids to CoA. Specifically, the proportion of arachidonic acidin fatty acid compositions produced by arachidonic acid-producing yeast(S. cerevisiae) expressing LPLAT5 and LPLAT6 according to preferredembodiments of the present invention increases, as further described inthe Examples below. In this case, LPLAT5 was found to be involved in theconversion from 18:1-CoA to 18:1-PL, conversion from 18:3(n-6)-PL to18:3(n-6)-CoA and conversion from DGLA-CoA to DGLA-PL, and LPLAT6 wasfound to be involved in the conversion from 18:3(n-6)-PL to18:3(n-6)-CoA and conversion from DGLA-CoA to DGLA-PL.

As described in the Examples below, LPLAT6 was also found to be involvedin the conversion from 18:3(n-6)-PL to 18:3(n-6)-CoA and conversion fromDGLA-CoA to DGLA-PL in M. alpina.

Therefore, the present invention also provides a method for using arecombinant vector to increase the proportion of arachidonic acid in thecompositional ratio of fatty acids in a host transformed with the vectoras compared with the proportion in the compositional ratio of fattyacids in a host that has not been transformed with the vector.

Food or Other Products Comprising Fatty Acid Compositions of the PresentInvention

The present invention also provides food products comprising the abovefatty acid compositions. The fatty acid compositions of the presentinvention can be routinely used to produce food products and industrialraw materials containing fats and oils (raw materials for cosmetics,pharmaceuticals (e.g., topical skin medicines), soaps, etc.) or forother purposes. Cosmetics (compositions) or pharmaceuticals(compositions) may be presented in any form including, but not limitedto, solution, paste, gel, solid, powder or the like. Food products mayalso be presented in the form of a pharmaceutical formulation such as acapsule, or a processed food such as a natural liquid diet, low residuediet, elemental diet, nutritional drink or enteral feeding formulacomprising a fatty acid composition of the present invention incombination with proteins, sugars, fats, trace elements, vitamins,emulsifiers, flavorings, etc.

Other examples of food products of the present invention include, butare not limited to, dietary supplements, health foods, functional foods,diets for children, modified milk for infants, modified milk forpremature infants, geriatric diets, etc. The food products as usedherein collectively refer to edible products in the form of solid,fluid, liquid or a mixture thereof.

Dietary supplements refer to food products fortified with specificnutritional ingredients. Health foods refer to food products known to behealthy or good for health, and include dietary supplements, naturalfoods, dietetic foods, etc. Functional foods refer to food products forsupplying nutritional ingredients having physiological controlfunctions, and may also be called foods for specified health use. Dietsfor children refer to food products intended for children up to about 6years of age. Geriatric diets refer to food products treated to easedigestion and absorption as compared with untreated foods. Modified milkfor infants refers to modified milk intended for children up to aboutone year of age. Modified milk for premature infants refers to modifiedmilk intended for premature infants of up to about 6 months of age.

These food products include natural foods such as meat, fish, nuts(treated with fats and oils); foods cooked with fats and oils such asChinese foods, Chinese noodles, soups; foods using fats and oils asheating media such as Tempura (deep-fried fish and vegitables),deep-fried foods coated in breadcrumbs, fried bean curd, Chinese friedrice, doughnuts, Karinto (Japanese fried dough cookies); fat- andoil-based food products or food products processed with fats and oilssuch as butter, margarine, mayonnaise, salad dressing, chocolate,instant noodles, caramel, biscuits, cookies, cake, ice cream; and foodproducts sprayed or coated with fats and oils during finishing such asrice crackers, hard biscuits, sweet bean paste bread. However, the foodproducts of the present invention are not limited to fat- andoil-containing foods, but also include processed agricultural foods suchas bread, noodles, cooked rice, sweets (candies, chewing gums, gummies,tablets, Japanese sweets), bean curd and processed products thereof;fermented foods such as Sake (Japanese rice wine), medicinal liquor,Mirin (sweet cooking sherry), vinegar, soy sauce and Miso (soy beanpaste); livestock food products such as yogurt, ham, bacon and sausage;processed seafood products such as Kamaboko (fish cake), Ageten(deep-fried fish cake) and Hanpen (puffy fish cake); and fruit drinks,soft drinks, sports drinks, alcoholic beverages, tea and the like.

Method for Evaluating or Selecting Strains Using Nucleic Acids EncodingLPLAT Proteins or LPLAT Proteins of the Present Invention

The present invention also provides methods for evaluating or selectinglipid-producing strains using nucleic acids encoding LPLAT proteins orLPLAT proteins of the present invention. The methods are specificallydescribed below.

(1) Evaluation Methods

One embodiment of the present invention is a method for evaluating alipid-producing strain using a nucleic acid encoding an LPLAT protein oran LPLAT protein of the present invention. The evaluation method of thepresent invention may comprise evaluating a lipid-producing test strainfor the above activity of the present invention using a primer or probedesigned on the basis of a nucleotide sequence of the present invention.General procedures for such an evaluation method are known and describedin, e.g., WO01/040514 or JP HEI 8-205900 A. This evaluation method isbriefly explained below.

First, the genome of a test strain is prepared. Any known preparationmethod can be used such as the Hereford method or potassium acetatemethod (see, e.g., Methods in Yeast Genetics, Cold Spring HarborLaboratory Press, p 130 (1990)).

A primer or probe is designed on the basis of a nucleotide sequence ofthe present invention, preferably SEQ ID NO: 1 or 6. The primer or probecan be designed from any region of the nucleotide sequence of thepresent invention using known procedures. The number of nucleotides in apolynucleotide used as a primer is typically 10 or more, preferably 15to 25. Typically, the number of nucleotides appropriate for a region tobe flanked by the primers is generally 300 to 2000.

The primer or probe prepared above is used to assess whether or not thegenome of the above test strain contains a sequence specific to thenucleotide sequence of the present invention. A sequence specific to thenucleotide sequence of the present invention may be detected using knownprocedures. For example, a polynucleotide comprising a part or all of asequence specific to the nucleotide sequence of the present invention ora polynucleotide comprising a nucleotide sequence complementary to theabove nucleotide sequence is used as one primer, and a polynucleotidecomprising a part or all of a sequence upstream or downstream of thissequence or a polynucleotide comprising a nucleotide sequencecomplementary to the above nucleotide sequence is used as the otherprimer to amplify the nucleic acid of the test strain by PCR or thelike, thereby determining the presence or absence of an amplifiedproduct, the molecular weight of the amplified product, etc.

PCR conditions suitable for the method of the present invention are notspecifically limited. The resulting reaction product, i.e., theamplified product can be separated by electrophoresis on agarose gel orthe like to determine the molecular weight of the amplified product.Thus, the above activity of the present invention of the test strain canbe predicted or evaluated by assessing whether or not the molecularweight of the amplified product is enough to cover a nucleic acidmolecule corresponding to a region specific to the nucleotide sequenceof the present invention. Moreover, the above activity of the presentinvention can be more accurately predicted or evaluated by analyzing thenucleotide sequence of the amplified product by the method describedabove or the like. The method for evaluating the above activity of thepresent invention is as described above.

Alternatively, the evaluation method of the present invention maycomprise culturing a test strain and determining the expression level ofan LPLAT protein encoded by a nucleotide sequence of the presentinvention such as SEQ ID NO: 1 or 6, thereby evaluating the test strainfor the above activity of the present invention. The expression level ofthe LPLAT protein can be determined by culturing the test strain underappropriate conditions and quantifying mRNA of the LPLAT protein or theprotein. Quantification of mRNA or the protein may be accomplished byusing known procedures. Quantification of mRNA may be accomplished by,for example, Northern hybridization or quantitative RT-PCR, whilequantification of the protein may be accomplished by, for example,Western blotting (Current Protocols in Molecular Biology, John Wiley &Sons 1994-2003).

(2) Selection Methods

Another embodiment of the present invention is a method for selecting alipid-producing strain using a nucleic acid encoding an LPLAT protein oran LPLAT protein of the present invention. The selection method of thepresent invention may comprise culturing test strains and determiningthe expression level of an LPLAT protein encoded by a nucleotidesequence of the present invention such as SEQ ID NO: 1 or 6 to select astrain having a desired expression level, whereby a strain having adesired activity can be selected. Alternatively, it may comprisepredetermining a type strain, separately culturing the type strain andtest strains, determining the above expression level in each strain, andcomparing the expression level between the type strain and each teststrain, whereby a desired strain can be selected. Specifically, a strainhaving a desired activity can be selected by culturing a type strain andtest strains under appropriate conditions, determining the expressionlevel in each strain, and selecting a test strain showing a higher orlower expression level than that of the type strain, for example. Thedesired activity may be assessed by determining the expression level ofthe LPLAT protein, as described above.

Alternatively, the selection method of the present invention maycomprise culturing test strains and selecting a strain showing a higheror lower level of the above activity of the present invention, whereby astrain having a desired activity can be selected. The desired activitymay be assessed by determining the expression level of the LPLATprotein, as described above.

Examples of test strains or type strains that can be used include forexample, but are not limited to, a strain transformed with the abovevector of the present invention, a strain with suppressed expression ofthe above nucleic acid of the present invention, a mutagenized strain, anaturally mutated strain, etc. Mutagenesis techniques include, but notlimited to, physical methods such as UV or radioactive irradiation, andchemical methods such as chemical treatments with EMS (ethylmethanesulfonate), N-methyl-N-nitrosoguanidine or the like (see, e.g.,Yasuji Oshima ed., Biochemistry Experiments vol. 39, ExperimentalProtocols for Yeast Molecular Genetics, pp. 67-75, Japan ScientificSocieties Press).

Strains used as type and test strains of the present invention include,but are not limited to, the lipid-producing fungi or yeast listed above.Specifically, the type and test strains may be a combination of anystrains belonging to different genera or species, and one or more teststrains may be used simultaneously.

The following examples further illustrate the present invention.However, it should be understood that the present invention is notlimited to the Examples below.

EXAMPLES Example 1 Genomic Analysis of M. alpina

M. alpina strain 1S-4 was inoculated into 100 ml of GY2:1 medium (2%glucose, 1% yeast extract, pH 6.0) and cultured with shaking for 2 daysat 28° C. The cells were harvested by filtration to prepare genomic DNAusing DNeasy (QIAGEN).

The nucleotide sequence of the genomic DNA was determined usingRoche454GS FLX Standard. This involved two runs of fragment librarysequence sequencing and three runs of mate pair library sequencing. Theresulting nucleotide sequences were assembled into 300 supercontigs.

Construction of cDNA Libraries

M. alpina strain 1S-4 was inoculated into 100 ml of a medium (1.8%glucose, 1% yeast extract, pH 6.0) and precultured for 3 days at 28° C.The total amount of the preculture was inoculated into 5 L of a medium(1.8% glucose, 1% soybean powder, 0.1% olive oil, 0.01% Adekanol, 0.3%KH₂PO₄, 0.1% Na₂SO₄, 0.05% CaCl₂.2H₂O, 0.05% MgCl₂.6H₂O, pH 6.0) in a 10L culture vessel (Able Co., Tokyo) and incubated with aeration andagitation under conditions of 300 rpm, 1 vvm, 26° C. for 8 days. Onincubation days 1, 2 and 3, glucose was added in amounts equivalent to2%, 2% and 1.5%, respectively. At each stage of incubation days 1, 2, 3,6 and 8, cells were harvested to prepare total RNA by the guanidinehydrochloride/CsCl method. Using an Oligotex-dT30<Super> mRNAPurification Kit (“dT30” disclosed as SEQ ID NO: 45) (Takara Bio Inc.),poly(A)⁺RNA was purified from the total RNA. A cDNA library at eachstage was constructed using a ZAP-cDNA GigapackIII Gold Cloning Kit(STRATAGENE).

Search for Homologs of SCL4 from S. cerevisiae

Supercontigs containing the sequences shown in SEQ ID NO: 5 and SEQ IDNO: 10 were identified by tblastn analysis of the amino acid sequencededuced from SLC4 (YOR175c) encoding an LPLAT of the MBOAT family of S.cerevisiae (PfamPFO3062) against the genomic nucleotide sequence of M.alpina 1S-4.

Preparation of a Probe

To clone cDNAs corresponding to SEQ ID NO: 5 and SEQ ID NO: 10, thefollowing primers were prepared (Table 2).

TABLE 2 Primers MaLPAAT5-1F(SEQ ID NO: 11) CTGTCTCCTTCCCAGAGGATCAGCMaLPAAT5-3R(SEQ ID NO: 12) ATAACCAAAGCGCAAGATCCATGGMaLPAAT6-2F(SEQ ID NO: 13) GTTGCCCACGTTGGCCGAGACGATCMaLPAAT6-3R(SEQ ID NO: 14) ATGGGTTCCGTGCCAGATCGCCAAG

The cDNA libraries were used as templates to perform PCR with ExTaq(Takara Bio Inc.) and the above primers in the following sets:MaLPAAT5-1F/MaLPAAT5-3R and MaLPAAT6-2F/MaLPAAT5-3R. The resulting DNAfragments were cloned using a TOPO-TA cloning kit (INVITROGEN) to give aplasmid containing nucleotides 195-931 of SEQ ID NO: 4 designatedpCR-LPLAT5-P and a plasmid containing nucleotides 766-1414 of SEQ ID NO:9 designated pCR-LPLAT6-P. Then, these plasmids were used as templatesto perform PCR with the above primers. ExTaq (Takara Bio Inc.) was usedfor the reaction, but a PCR labeling mix (Roche Diagnostics) was usedinstead of the dNTP mix included in the kit to prepare a probe labeledwith digoxigenin (DIG) from the amplified DNA. This probe was used toscreen the cDNA libraries.

Hybridization conditions are as follows.

Buffer: 5×SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5), 50% formamide;Temperature: 42° C. (overnight);Washing conditions: 3 times in a solution of 0.2×SSC, 0.1% SDS (65° C.)for 20 minutes. Detection was accomplished by using a DIG nucleic aciddetection kit (Roche Diagnostics). Plasmids were excised by in vivoexcision from phage clones obtained by screening to yield each plasmidDNA. The plasmid having the longest insert among those obtained byscreening with LPLAT5 probe 1 was designated pB-LPLAT5, and the plasmidhaving the longest insert among those obtained by screening with LPLAT6probe 1 was designated pB-LPLAT6. The nucleotide sequence of the insertof plasmid pB-LPLAT5, i.e., the cDNA of LPLAT5 was SEQ ID NO: 4, whilethe nucleotide sequence of the insert of plasmid pB-LPLAT6, i.e., thecDNA of LPLAT6 was SEQ ID NO: 9.

Sequence Analysis

The cDNA sequence of LPLAT5, i.e., SEQ ID NO: 4 contained a CDSconsisting of nucleotides 161-1693 (SEQ ID NO: 3) and an ORF consistingof nucleotides 161-1690 (SEQ ID NO: 1). The cDNA sequence of LPLAT5 andits deduced amino acid sequence were described in FIG. 2.

On the other hand, the cDNA sequence of LPLAT6, i.e., SEQ ID NO: 9contained a CDS consisting of nucleotides 38-1759 (SEQ ID NO: 8) and anORF consisting of nucleotides 38-1756 (SEQ ID NO: 6). The cDNA sequenceof LPLAT6 and its deduced amino acid sequence were described in FIG. 3.

SEQ ID NO: 1 and SEQ ID NO: 6 were subjected to homology analysis usingBLASTX against amino acid sequences deposited in GENEBANK. The aminoacid sequence deduced from SEQ ID NO: 1 showed homology to LPLAThomologs from fungi, while the amino acid sequence deduced from SEQ IDNO: 6 showed homology to LPLAT homologs from animals. The amino acidsequences showing the lowest E-value or the highest identity to eachsequence were as follows. The nucleotide sequence identity and aminoacid sequence identity of the sequence showing the highest identity tothe ORF of each sequence were determined by clustalW and also reportedbelow.

SEQ ID NO: 1 had 43.2% nucleotide sequence identity and 33.3% amino acidsequence identity in ORF to a lysophospholipid acyltransferase homologfrom Schizosaccharomyces pombe (GI:161085648). On the other hand, SEQ IDNO: 6 had 41.2% nucleotide sequence identity and 28.6% amino acidsequence identity in ORF to a putative protein from Xenopus laevis(GI:56788919). The nucleotide sequence identity and amino acid sequenceidentity in ORF between LPLAT and LPLAT6 are 40.0% and 19.1%,respectively.

The genomic sequences containing the CDS of LPLAT5 (SEQ ID NO: 3) andthe CDS of LPLAT6 (SEQ ID NO: 8) were described in SEQ ID NO: 5 and SEQID NO: 10, respectively. SEQ ID NO: 5 contained two introns and exonscorresponding to nucleotides 1-314, 461-587, and 668-1759. On the otherhand, SEQ ID NO: 10 contained one intron and exons corresponding tonucleotides 1-1095 and 1318-1944. FIG. 4 depicts the alignment betweenthe genomic sequence and ORF sequence of LPLAT5, and FIG. 5 depicts thealignment between the genomic sequence and ORF sequence of LPLAT6.

Example 2

Construction of Yeast Expression Vectors

In order to express LPLAT5 and LPLAT6 in yeast, vectors were constructedas follows.

Using pBLPLAT5 as a template, PCR was performed with ExTaq (Takara Bio)and

primer Eco-MaLPLAT5-F(SEQ ID NO: 15): GAATTCATGCTAAACTCATTCTTCGGGGACGCand primer Xho-MaLPLAT5-R(SEQ ID NO: 16):CTCGAGTTACAGCGTCTTGATTTTAACTGCAGC.

The resulting DNA fragments were TA-cloned using a TOPO-TA cloning Kit(INVITROGEN), and the nucleotide sequence of the insert was determinedto give a plasmid having a correct nucleotide sequence designatedpCR-LPLAT5. A DNA fragment of about 1.6 kb obtained by digesting thisplasmid with restriction endonucleases EcoRI and XhoI was inserted intothe EcoRI-SalI site of a yeast expression vector pYE22m (Appl.Microbiol. Biotechnol., 30, 515-520, 1989) to generate plasmidpYE-MALPLAT5.

On the other hand, a DNA fragment of 1.9 kb obtained by digestingpBLPLAT6 with restriction endonucleases EcoRI and KpnI was inserted intothe EcoRI-KpnI site of the yeast-expressing vector pYE22m to generateplasmid pYE-LPLAT6.

Expression in Arachidonic Acid-Producing Yeast

(1) Breeding of arachidonic acid-producing yeast To breed arachidonicacid-producing yeast (S. cerevisiae), the following plasmids wereconstructed.

First, PCR was performed using cDNA prepared from M. alpina strain 1S-4as a template with ExTaq and the following primer set: Δ12-f/Δ12-r,Δ6-f/Δ6-r, GLELO-f/GLELO-r or Δ5-f/Δ5-r to amplify the Δ12 fatty aciddesaturase gene, Δ6 fatty acid desaturase gene, GLELO fatty acidelongase gene and Δ5 fatty acid desaturase gene of M. alpina strain1S-4.

Δ12-f: (SEQ ID NO: 17) TCTAGAATGGCACCTCCCAACACTATTG Δ12-r:(SEQ ID NO: 18) AAGCTTTTACTTCTTGAAAAAGACCACGTC Δ6-f: (SEQ ID NO: 19)TCTAGAATGGCTGCTGCTCCCAGTGTGAG Δ6-r: (SEQ ID NO: 20)AAGCTTTTACTGTGCCTTGCCCATCTTGG GLELO-f: (SEQ ID NO: 21)TCTAGAATGGAGTCGATTGCGCAATTCC GLELO-r: (SEQ ID NO: 22)GAGCTCTTACTGCAACTTCCTTGCCTTCTC Δ5-f: (SEQ ID NO: 23)TCTAGAATGGGTGCGGACACAGGAAAAACC Δ5-r: (SEQ ID NO: 24)AAGCTTTTACTCTTCCTTGGGACGAAGACC.

These were cloned using a TOPO-TA-cloning Kit. The nucleotide sequenceswere identified, and the clones containing the nucleotide sequences weredesignated as plasmids pCR-MAΔ12DS (containing the nucleotide sequenceof SEQ ID NO: 25), pCR-MAΔ6DS (containing the nucleotide sequence of SEQID NO: 26), pCR-MAGLELO (containing the nucleotide sequence of SEQ IDNO: 27), and pCR-MAΔ5DS (containing the nucleotide sequence of SEQ IDNO: 28).

A DNA fragment of about 1.2 kb obtained by digesting plasmid pURA34 (JP2001-120276 A) with restriction endonuclease HindIII was inserted intothe HindIII site of a vector obtained by digesting the vector pUC18 withrestriction endonucleases EcoRI and SphI followed by blunt-ending andself-ligating to generate a clone designated pUC-URA3 with the EcoRIsite of the vector at the 5′-end of URA3. A DNA fragment of about 2.2 kbobtained by digesting YEp13 with restriction endonucleases SalI and XhoIwas inserted into the SalI site of the vector pUC18 to generate a clonedesignated pUC-LEU2 with the EcoRI site of the vector at the 5′-end ofLEU2.

Then, a DNA fragment of about 1.2 kbp obtained by digesting plasmidpCR-MAΔ12DS with restriction endonuclease HindIII followed byblunt-ending and further digesting it with restriction endonuclease XbaIwas ligated to a DNA fragment of about 6.6 kbp obtained by digesting thevector pESC-URA (STRATAGENE) with restriction endonuclease SacI followedby blunt-ending and further digesting it with restriction endonucleaseSpeI to generate plasmid pESC-U-Δ12. A DNA fragment of about 1.6 kbpobtained by digesting plasmid pCR-MAΔ6DS with restriction endonucleaseXbaI followed by blunt-ending and further digesting it with restrictionendonuclease HindIII was ligated to a DNA fragment of about 8 kbpobtained by digesting plasmid pESC-U-Δ12 with restriction endonucleaseSalI followed by blunt-ending and further digesting it with restrictionendonuclease HindIII to generate plasmid pESC-U-Δ12:Δ6. A fragment ofabout 4.2 kb obtained by partially digesting this with restrictionendonuclease PvuII was inserted into the SmaI site of pUC-URA3 togenerate plasmid pUC-URA-Δ12:Δ6.

A DNA fragment of about 0.95 kbp obtained by digesting plasmidpCR-MAGLELO with restriction endonucleases XbaI and SacI was ligated toa DNA fragment of about 7.7 kbp obtained by digesting the vectorpESC-LEU (STRATAGENE) with restriction endonucleases XbaI and SacI togenerate plasmid pESC-L-GLELO. A DNA fragment of about 1.3 kbp obtainedby digesting plasmid pCR-MAΔ5DS with restriction endonuclease XbaIfollowed by blunt-ending and further digesting it with restrictionendonuclease HindIII was ligated to a DNA fragment of about 8.7 kbpobtained by digesting plasmid pESC-L-GLELO with restriction endonucleaseApaI followed by blunt-ending and further digesting it with restrictionendonuclease HindIII to generate plasmid pESC-L-GLELO:Δ5. A fragment ofabout 3.2 kb obtained by digesting this with restriction endonucleasePvuII was inserted into the SmaI site of pUC-LEU2 to generate plasmidpUC-LEU-GLELO:Δ5. S. cerevisiae strain YPH499 (STRATAGENE) wasco-transformed with plasmid pUC-URA-Δ12:Δ6 and plasmid pUC-LEU-GLELO:Δ5.Transformed strains were selected by viability on SC-Leu, Ura agarmedium (2% agar) containing, per liter, 6.7 g Yeast nitrogen base w/oamino acids (DIFCO), 20 g glucose and 1.3 g amino acid powder (a mixtureof 1.25 g adenine sulfate, 0.6 g arginine, 3 g aspartic acid, 3 gglutamic acid, 0.6 g histidine, 0.9 g lysine, 0.6 g methionine, 1.5 gphenylalanine, 11.25 g serine, 0.9 g tyrosine, 4.5 g valine, 6 gthreonine and 1.2 g tryptophan). Random one of the selected strains wasdesignated as ARA3-1 strain. This strain can produce arachidonic acid byexpressing the Δ12 fatty acid desaturase gene, Δ6 fatty acid desaturasegene, GLELO fatty acid elongase gene, and Δ5 fatty acid desaturase genefrom the GALL/10 promoter upon cultivation in a galactose-containingmedium.

(2) Transformation of Arachidonic Acid-Producing Yeast

ARA3-1 strain was transformed with plasmids pYE22m, pYE-MALPLAT5, andpYE-MALPLAT6. Transformed strains were selected by viability on SC-Trp,Leu, Ura agar medium (2% agar) containing, per liter, 6.7 g Yeastnitrogen base w/o amino acids (DIFCO), 20 g glucose and 1.3 g amino acidpowder (a mixture of 1.25 g adenine sulfate, 0.6 g arginine, 3 gaspartic acid, 3 g glutamic acid, 0.6 g histidine, 0.9 g lysine, 0.6 gmethionine, 1.5 g phenylalanine, 11.25 g serine, 0.9 g tyrosine, 4.5 gvaline, and 6 g threonine).

Random three strains transformed with each plasmid were used for thesubsequent cultivation. In Tables 3-8 below, control represents strainstransformed with plasmid pYE22m, LPLAT5 represents strains transformedwith plasmid pYE-MALPLAT5, and LPLAT6 represents strains transformedwith plasmid pYE-MALPLAT6.

(3) Cultivation in a Fatty Acid Free Medium

The above transformed strains were cultured with shaking in 10 ml ofSC-Trp, Leu, Ura liquid medium at 30° C. for 1 day, and 1 ml of thecultures were incubated with shaking in 10 ml of SG-Trp, Leu, Ura liquidmedium containing, per liter, 6.7 g Yeast nitrogen base w/o amino acids(DIFCO), 20 g galactose and 1.3 g amino acid powder (a mixture of 1.25 gadenine sulfate, 0.6 g arginine, 3 g aspartic acid, 3 g glutamic acid,0.6 g histidine, 0.9 g lysine, 0.6 g methionine, 1.5 g phenylalanine,11.25 g serine, 0.9 g tyrosine, 4.5 g valine, and 6 g threonine) at 15°C. for 6 days. The cells were harvested, washed with water and thenlyophilized and subjected to fatty acid analysis.

The results are shown in Table 3.

TABLE 3 Compositional ratio of fatty acids in yeast cells expressingeach gene (%) - cultured in a fatty acid-free medium - Control LPLAT5LPLAT6 16:0 22.16 ± 0.42  20.88 ± 0.13  20.38 ± 0.13  16:1 28.79 ± 0.55 30.81 ± 0.32  30.22 ± 0.31  18:0 10.35 ± 0.23  9.98 ± 0.08 9.81 ± 0.1118:1 20.28 ± 0.30  17.08 ± 0.17  20.81 ± 0.21  18:2 7.61 ± 0.05 9.16 ±0.15 8.10 ± 0.09 18:3 (n-6) 0.47 ± 0.03 0.18 ± 0.01 0.11 ± 0.02 DGLA0.46 ± 0.01 0.40 ± 0.02 0.00 ± 0.00 ARA 0.38 ± 0.02 0.58 ± 0.03 0.89 ±0.03 other 9.51 ± 0.94 11.03 ± 0.77  9.68 ± 0.15 mean ± SD

Based on the results in Table 3, the conversion of a fatty acid toanother fatty acid in the arachidonic acid synthetic pathway wasdetermined. For example, the conversion of 18:2→48:3(n-6) is determinedas follows:

Conversion=(18:3(n-6)+DGLA+ARA)/(18:2+18:3(n-6)+DGLA+ARA)×100

The results are shown in Table 4.

TABLE 4 Conversions of fatty acids in the arachidonic acid biosyntheticpathway (%) - cultured in a fatty acid-free medium - Control LPLAT5LPLAT6 18:1→18:2 29.63 ± 0.28 36.09 ± 0.39 28.29 ± 0.40 18:2→18:3(n-6)14.63 ± 0.55 11.33 ± 0.35 10.99 ± 0.42 18:3(n-6)→DGLA 64.15 ± 0.85 84.50± 0.91 88.81 ± 1.81 DGLA→ARA 45.17 ± 1.61 59.02 ± 0.30 100.00 ± 0.00 mean ± SD

As shown in Tables 3 and 4, the proportion of arachidonic acid to totalfatty acids increased 1.5-fold in the LPLAT5-expressing strains and2.3-fold in the LPLAT6-expressing strains as compared with the control.The conversions of fatty acids in the arachidonic acid biosyntheticpathway were reviewed, revealing that the conversions of 18:1→48:2,18:3(n-6)→DGLA, and DGLA→ARA increased in the LPLAT5-expressing strainswhile the conversion of DGLA→ARA remarkably increased in theLPLAT6-expressing strains. These conversions required acyl transfer fromacyl-CoA to phospholipids or from phospholipids to CoA as shown in FIG.1, suggesting that LPLAT5 and LPLAT6 are involved in these conversions.

(4) Cultivation in a Medium Containing Linoleic Acid

The transformed strains were cultured with shaking in 10 ml of SC-Trp,Leu, Ura liquid medium at 30° C. for 1 day, and 1 ml of the cultureswere inoculated into 10 ml of SG-Trp, Leu, Ura liquid medium containing5 mg/ml linoleic acid and 0.1% Triton X-100 and incubated with shakingat 15° C. for 6 days. Cells were harvested, washed with water and thenlyophilized and subjected to fatty acid analysis. The results are shownin Table 5.

TABLE 5 Compositional ratio of fatty acids in yeast cells expressingeach gene (%) - cultured in a medium containing linoleic acid - ControlLPLAT5 LPLAT6 16:0 21.30 ± 0.44  19.20 ± 0.10  21.45 ± 0.22  16:1 17.33± 0.56  17.98 ± 0.10  18.69 ± 0.20  18:0 7.82 ± 0.43 7.74 ± 0.05 8.05 ±0.15 18:1 10.12 ± 0.26  9.21 ± 0.03 10.69 ± 0.17  18:2 36.05 ± 0.44 39.51 ± 0.05  34.53 ± 0.27  18:3(n-6) 0.69 ± 0.06 0.35 ± 0.07 0.09 ±0.06 DGLA 0.29 ± 0.02 0.29 ± 0.01 0.04 ± 0.09 ARA 0.12 ± 0.02 0.24 ±0.01 0.42 ± 0.01 other 6.27 ± 0.25 5.50 ± 0.15 6.04 ± 0.29 mean ± SD

Based on the results in Table 5, the conversion of a fatty acid toanother fatty acid in the arachidonic acid synthetic pathway wasdetermined. The results are shown in Table 6.

TABLE 6 Conversions of fatty acids in the arachidonic acid biosyntheticpathway (%) Control LPLAT5 LPLAT6 18:2→18:3(n-6)  2.97 ± 0.12  2.15 ±0.22 1.57 ± 0.37 18:3(n-6)→DGLA 37.65 ± 2.58 60.49 ± 4.53 85.08 ± 10.00DGLA→ARA 29.66 ± 3.51 45.43 ± 1.86 92.72 ± 14.56 mean ± SD

As shown in Table 5, the proportion of arachidonic acid to total fattyacids increased 2-fold in the LPLAT5-expressing strains and 3.5-fold inthe LPLAT6-expressing strains as compared with the control. In theLPLAT5-expressing strains, the proportion of linoleic acid addedincreased as compared with the control. The conversions of fatty acidsin the arachidonic acid biosynthetic pathway (Table 6) were reviewed,revealing that the conversions of 18:3(n-6)→DGLA and DGLA→ARA increasedin both LPLAT5-expressing strains and LPLAT6-expressing strains,especially remarkably increased in the LPLAT6-expressing strains.

(5) Cultivation in a Medium Containing γ-Linolenic Acid

The transformed strains were cultured with shaking in 10 ml of SC-Trp,Leu, Ura liquid medium at 30° C. for 1 day, and 1 ml of the cultureswere inoculated into 10 ml of SG-Trp, Leu, Ura liquid medium containing5 mg/ml γ-linolenic acid and 0.1% Triton X-100 and incubated withshaking at 15° C. for 6 days. Cells were harvested, washed with waterand then lyophilized and subjected to fatty acid analysis. The resultsare shown in Table 7.

TABLE 7 Compositional ratio of fatty acids in yeast cells expressingeach gene (%) - cultured in a medium containing γ-linolenic acid -Control LPLAT5 LPLAT6 16:0 20.97 ± 0.24  17.59 ± 0.06  22.02 ± 0.09 16:1 16.11 ± 1.02  17.28 ± 0.23  16.17 ± 0.37  18:0 8.54 ± 0.06 7.78 ±0.08 9.59 ± 0.07 18:1 9.03 ± 0.86 8.80 ± 0.04 9.46 ± 0.17 18:2 4.57 ±0.11 5.10 ± 0.04 5.02 ± 0.10 18:3(n-6) 20.36 ± 1.67  25.28 ± 0.26  17.97± 0.78  DGLA 10.88 ± 0.29  9.60 ± 0.05 1.86 ± 0.07 ARA 4.74 ± 0.09 4.82± 0.04 13.50 ± 0.22  other 4.81 ± 0.11 3.75 ± 0.04 4.42 ± 0.06 mean ± SD

Based on the results in Table 7, the conversion of a fatty acid toanother fatty acid in the arachidonic acid synthetic pathway wasdetermined (Table 8).

TABLE 8 Conversions of fatty acids downstream of γ-linolenic acid in thearachidonic acid biosynthetic pathway (%) - cultured in a mediumcontaining γ-linolenic acid - Control LPLAT5 LPLAT6 18:3(n-6)→DGLA 43.25± 1.29 36.33 ± 0.27 46.10 ± 1.14 DGLA→ARA 31.70 ± 2.70 33.44 ± 0.2687.91 ± 0.54 mean ± SD

As shown in Table 7, the proportion of γ-linolenic acid added to totalfatty acids increased in the LPLAT5-expressing strains. However, theproportions of the downstream products dihomo-γ-linolenic acid andarachidonic acid did not increase (Table 8). In contrast, the proportionof arachidonic acid to total fatty acids increased 2.8-fold as comparedwith the control and the conversion of DGLA→ARA significantly increasedin the LPLAT6-expressing strains (Table 8).

These results show that LPLAT5 and LPLAT6 can increase the conversionsof fatty acids requiring acyl transfer from acyl-CoA to phospholipids orfrom phospholipids to CoA. The involvement of LPLAT5 in the conversionfrom 18:1-CoA to 18:1-PL, conversion from 18:3(n-6)-PL to 18:3(n-6)-CoAand conversion from DGLA-CoA to DGLA-PL was suggested. On the otherhand, the involvement of LPLAT6 in the conversion from 18:3(n-6)-PL to18:3(n-6)-CoA and conversion from DGLA-CoA to DGLA-PL was suggested.

Example 3 Functional Analysis of LPLAT6 in M. alpina

Construction of Mortierella Expression Vectors

The following oligonucleotides were synthesized for use as adapters.

A-1: (SEQ ID NO: 29) GATCCGGCGCGCCGCGGCCGCTCTAGAGTCGACGGCGCGCCA A-2:(SEQ ID NO: 30) AGCTTGGCGCGCCGTCGACTCTAGAGCGGCCGCGGCGCGCCG.

A-1 and A-2 were annealed and ligated to a fragment obtained bydigesting the plasmid pUC18 with restriction endonucleases EcoRI andHindIII to generate pUC18-R.

Using genomic DNA or a plasmid prepared from M. alpina strain 1S-4 as atemplate, each DNA fragment was amplified by PCR using ExTaq (TakaraBio) with the following primer set and cloned using TOPO-TA cloning Kit(Invitrogen).

Specifically, genomic DNA was used as a template to amplify genomic DNAof about 2 kbp containing the URA5 gene using the primer set:

primer URA5g-F1: (SEQ ID NO: 31) GTCGACCATGACAAGTTTGC, andprimer URA5g-R1: (SEQ ID NO: 32) GTCGACTGGAAGACGAGCACG;to amplify the GAPDH promoter of about 0.9 kbp using the primer set:

primer GAPDHp-F1: (SEQ ID NO: 33) GTCGACGATCACGTCGGGTGATGAGTTG, andprimer GAPDHp-R1: (SEQ ID NO: 34) TCTAGAGATGTTGAATGTGTGGTGTGTG;and to amplify the GAPDH terminator of about 0.5 kbp using the primerset:

primer GAPDHt-F1: (SEQ ID NO: 35) GCGGCCGCTAAGAAAAGGGAGTGAATCGC, andprimer GAPDHt-R1: (SEQ ID NO: 36) GGATCCGGCGCGCCGATCCATGCACGGGTCCTTCTC.

Plasmid pB-LPLAT6 was used as a template to amplify the CDS of about 1.6kbp of the LPLAT6 gene using the primer set:

primer XbaI-LPLAT6-F1: (SEQ ID NO: 37) TCTAGAATGGAGGCACTCTTGCACCAGG, andprimer NotI-LPLAT6-R1: (SEQ ID NO: 38) GCGGCCGCTTACTCAGTCTTGACAGACTTG;and to amplify a 3′-fragment of about 0.7 kbp of the CDS of the LPLAT6gene using the primer set:

primer EcoRV-LPLAT6-F2: (SEQ ID NO: 39) GATATCGGGTAAAGCCTTCCTGGAACG, andprimer XbaI-LPLAT6-R2: (SEQ ID NO: 40)TCTAGATTACTCAGTCTTGACAGACTTGGATCG.Likewise, plasmid pCR-MAΔ5DS was used as a template to amplify the CDSof about 1.3 kbp of the Δ5 fatty acid desaturase gene using the primerset:

primer XbaI-Δ5DS-F1: (SEQ ID NO: 41) TCTAGAATGGGTGCGGACACAGGAAAAAC, andprimer NotI-Δ5DS-R1: (SEQ ID NO: 42) GCGGCCGCTTACTCTTCCTTGGGACGAAG;and to amplify a 3′-fragment of about 0.5 kbp of the CDS of the Δ5 fattyacid desaturase gene using the primer set:

primer NdeI-Δ5DS-R2: (SEQ ID NO: 43) TCTAGATTACTCTTCCTTGGGACGAAG, andprimer XbaI-Δ5DS-F2: (SEQ ID NO: 44) CATATGCATCCAGGACATCAACATCTTG.

Into the restriction endonuclease EcoRI/NotI sites of plasmid pUC18-Rwas inserted a fragment excised with the same restriction endonucleasesfrom the GAPDH terminator to generate plasmid pUC-GAPDHt. Subsequently,plasmid pUC-GAPDHt was cleaved with restriction endonucleases XbaI andSalI, and a fragment excised with the same restriction endonucleasesfrom the GAPDH promoter was inserted to generate plasmid pUC-GAPDHpt.Plasmid pUC-GAPDHpt was cleaved with restriction endonuclease SalI, anda fragment cleaved with the same restriction endonuclease from thegenomic DNA containing the URA5 gene was inserted. The orientations ofthe inserts were confirmed and a vector containing the URA5 geneinserted in the same orientation as that of the restriction endonucleasesites EcoRI→HindIII was selected and designated as plasmid pDUraRSC.

Plasmid pDUraRSC was cleaved with restriction endonucleases XbaI andNotI, and a DNA fragment excised with the same restriction endonucleasesfrom the CDS of the LPLAT6 gene was inserted to generate plasmidpDUraRSC-LPLAT6. A DNA fragment of about 7 kbp obtained by cleavingplasmid pDUraRSC-LPLAT6 with restriction endonucleases EcoRV and XbaIwas ligated to a DNA fragment excised with the same restrictionendonucleases from the 3′-fragment of about 0.7 kbp of the CDS of theLPLAT6 gene to generate plasmid pDUraRSC-LPLAT6-RNAi.

Construction of Vectors with Suppressed Expression of Δ5DS (RNAi)

Plasmid pDUraRSC was cleaved with restriction endonucleases XbaI andNotI, and a DNA fragment excised with the same restriction endonucleasesfrom the CDS of the Δ5 fatty acid desaturase gene to generate plasmidpDUraRSC-Δ5DS. A DNA fragment of about 1.2 kbp obtained by cleavingplasmid pDUraRSC-Δ5DS with restriction endonucleases EcoRI and NdeI wasligated to a DNA fragment of about 5.5 kbp obtained by cleaving it withrestriction endonucleases XbaI and EcoRI and a fragment excised withrestriction endonucleases NdeI and XbaI from the 3′-fragment of about0.5 kbp of the CDS of the Δ5 fatty acid desaturase gene to generateplasmid pDUraRSC-Δ5DS-RNAi.

Acquisition of Transformed M. alpina Strains

An uracil-auxotrophic strain Δura-3 derived from M. alpina according toa method described in a patent document (WO2005/019437 entitled “Methodof Breeding Lipid-Producing Fungus”) in plasmid pDUraRSC-LPLAT6-RNAi orplasmid pDUraRSC-Δ5DS-RNAi was used as a host and transformed by theparticle delivery method. SC agar medium (0.5% Yeast Nitrogen Base w/oAmino Acids and Ammonium Sulfate (Difco), 0.17% ammonium sulfate, 2%glucose, 0.002% adenine, 0.003% tyrosine, 0.0001% methionine, 0.0002%arginine, 0.0002% histidine, 0.0004% lysine, 0.0004% tryptophan, 0.0005%threonine, 0.0006% isoleucine, 0.0006% leucine, 0.0006% phenylalanine,and 2% agar) was used for selecting transformed strains.

Evaluation of Transformed M. alpina Strains

About 50 strains transformed with each plasmid were inoculated into 4 mlof GY medium (2% glucose, 1% yeast extract, pH 6.0) and cultured withshaking at 28° C. for 4 days. At the end of the cultivation, cells wereharvested by filtration and lyophilized. A part of the lyophilized cells(about 10-20 mg) were collected, and fatty acids in the cells wereconverted into methyl esters using methanolic HCl, then extracted withhexane, and hexane was distilled off and the residue was subjected tofatty acid analysis by gas chromatography. Among the strains transformedwith the different plasmids, those having a higher proportion ofdihomo-γ-linolenic acid than the proportion of arachidonic acid, i.e.,LPLAT6-D#6 (transformed with plasmid pDUraRSC-LPLAT6-RNAi) and Δ5DS-D#45(transformed with plasmid pDUraRSC-Δ5DS-RNAi) were selected.

These two strains and a control (wild-type M. alpina strain 1S-4) werecultured with shaking in 4 ml of GY medium at 28° C. for 4 days. At theend of the cultivation, cells were harvested by filtration andlyophilized. A part of the lyophilized cells (about 10-20 mg) werecollected, and mechanically disrupted. The cells were maintained in 4 mlof chloroform-methanol (2:1) at 70° C. for 1 hour with intermittentstirring, and then centrifuged to collect the supernatant. The remainingcells were maintained in another 4 ml of chloroform-methanol (2:1) at70° C. for 1 hour with intermittent stirring, and then centrifuged tocollect the supernatant, which was combined with the previoussupernatant. Lipids were dried in a SpeedVac centrifuge concentrator,and dissolved in 5 ml of chloroform. One ml of the solution was dried inthe same manner as described above, and fatty acids were converted intomethyl esters using methanolic HCl and subjected to fatty acid analysis.On the other hand, 2 ml of the solution in chloroform was also dried inthe same manner as described above, and dissolved in a small amount ofchloroform and the total amount of the solution was subjected tothin-layer chromatography as follows. Lipids were fractionated bythin-layer chromatography on silica gel 60 plates (Merck), eluting withhexane:diethyl ether:acetic acid of 70:30:1. The plates were sprayedwith an aqueous solution containing 0.015% Primuline, 80% acetone(Primuline solution), and lipids were visualized by UV irradiation,whereby triacylglycerol (TG) fractions and phospholipid (PL) fractionswere marked with a pencil and the silica gel in the marked areas wasscraped off and collected in test tubes. Fatty acids were converted intomethyl esters using methanolic HCl and subjected to fatty acid analysisby gas chromatography. Thus, fatty acids were converted into methylesters by a reaction with 1 ml of dichloromethane and 2 ml of 10%methanolic HCl at 50° C. for 3 hours. Then, 4 ml of hexane and 1 ml ofwater were added and the solution was vigorously stirred and thencentrifuged and the upper layer was collected. The solvent was distilledoff in a SpeedVac and the residue was dissolved in acetonitrile andsubjected to fatty acid analysis by gas chromatography. The results areshown in FIGS. 6-8.

FIG. 6 shows the composition ratio of polyunsaturated fatty acids intotal lipids extracted with chloroform-methanol (2:1). In contrast tothe control containing a high proportion of arachidonic acid, LPLAT6-D#6strain and Δ5DS-D#45 strain showed comparable proportions ofdihomo-γ-linolenic acid and arachidonic acid because of the inhibitionof the conversion from dihomo-γ-linolenic acid to arachidonic acid. FIG.7 shows the composition ratio of polyunsaturated fatty acids intriacylglycerols constituting a major portion of lipids in cells.Similarly to the composition ratio in total lipids in cells, LPLAT6-D#6strain and Δ5DS-D#45 strain showed a higher proportion ofdihomo-γ-linolenic acid as compared with the control. However, the fattyacid composition ratio in phospholipid fractions shown in FIG. 8differed greatly between LPLAT6-D#6 strain and Δ5DS-D#45 strain.Specifically, Δ5DS-D#45 strain showed a high proportion ofdihomo-γ-linolenic acid, while LPLAT6-D#6 strain showed a highproportion of arachidonic acid but behind the control and also showed ahigh proportion of γ-linolenic acid as compared with the control andΔ5DS-D#45 strain.

The biosynthetic pathway of arachidonic acid in M. alpina is presumed toproceed as shown in FIG. 1. The experiments described above alsostrongly suggested that the Δ5 fatty acid desaturase acts on DGLA-PL toproduce arachidonic acid. In contrast, 18:3(n-6)-PL accumulated in thestrains with suppressed expression of LPLAT6. The proportion of DGLA inTG fractions increased, but no significant increase of the proportion ofDGLA was observed in PL fractions. These results strongly suggested thatLPLAT6 is responsible for the conversion of 18:3(n-6)-PL to18:3(n-6)-CoA and the conversion of DGLA-CoA to DGLA-PL in M. alpina.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 11: primer

SEQ ID NO: 12: primer

SEQ ID NO: 13: primer

SEQ ID NO: 14: primer

SEQ ID NO: 15: primer

SEQ ID NO: 16: primer

SEQ ID NO: 17: primer

SEQ ID NO: 18: primer

SEQ ID NO: 19: primer

SEQ ID NO: 20: primer

SEQ ID NO: 21: primer

SEQ ID NO: 22: primer

SEQ ID NO: 23: primer

SEQ ID NO: 24: primer

SEQ ID NO: 29: adapter A-1

SEQ ID NO: 30: adapter A-2

SEQ ID NO: 31: primer URA5g-F1

SEQ ID NO: 32: primer URA5g-R1

SEQ ID NO: 33: primer GAPDHp-F1

SEQ ID NO: 34: primer GAPDHp-R1

SEQ ID NO: 35: primer GAPDHt-F1

SEQ ID NO: 36: primer GAPDHt-R1

SEQ ID NO: 37: primer XbaI-LPLAT6-F1

SEQ ID NO: 38: primer NotI-LPLAT6-R1

SEQ ID NO: 39: primer EcoRV-LPLAT6-F2

SEQ ID NO: 40: primer XbaI-LPLAT6-R2

SEQ ID NO: 41: primer XbaI-Δ5DS-F1

SEQ ID NO: 42: primer NotI-Δ5DS-R1

SEQ ID NO: 43: primer NdeI-Δ5DS-F1

SEQ ID NO: 44: primer XbaI-Δ5DS-R1

What is claimed is:
 1. A cDNA or recombinant vector comprising a nucleicacid of any one of (a)-(e) below: (a) a nucleic acid that comprises anucleotide sequence encoding a protein consisting of an amino acidsequence with deletion, substitution or addition of one to 50 aminoacids in the amino acid sequence shown in SEQ ID NO: 2, and havinglysophospholipid acyltransferase activity; (b) a nucleic acid thathybridizes under hybridization conditions of 0.1×SSC-1×SSC at 60° C.-65°C. and washing conditions of 0.2×SSC-2×SSC at 50° C.-68° C. to a nucleicacid consisting of a nucleotide sequence full length complement to thenucleotide sequence consisting of SEQ ID NO: 1 and that comprises anucleotide sequence encoding a protein having lysophospholipidacyltransferase activity; (c) a nucleic acid that comprises a nucleotidesequence sharing an identity of 90% or more with the nucleotide sequenceconsisting of SEQ ID NO: 1 and encoding a protein havinglysophospholipid acyltransferase activity; (d) a nucleic acid thatcomprises a nucleotide sequence encoding a protein consisting of anamino acid sequence sharing an identity of 90% or more with the aminoacid sequence consisting of SEQ ID NO: 2 and having lysophospholipidacyltransferase activity; and (e) a nucleic acid that hybridizes underhybridization conditions of 0.1×SSC-1×SSC at 60° C.-65° C. and washingconditions of 0.2×SSC-2×SSC at 50° C.-68° C. to a nucleic acidconsisting of a nucleotide sequence full length complement to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 and that comprises a nucleotide sequenceencoding a protein having lysophospholipid acyltransferase activity. 2.The cDNA or recombinant vector of claim 1, which is (a), (c), or (d)below: (a) a nucleic acid that comprises a nucleotide sequence encodinga protein consisting of an amino acid sequence with deletion,substitution or addition of 1-25 amino acids in the amino acid sequenceshown in SEQ ID NO: 2, and having lysophospholipid acyltransferaseactivity; (c) a nucleic acid that comprises a nucleotide sequencesharing an identity of 95% or more with the nucleotide sequenceconsisting of SEQ ID NO: 1 and encoding a protein havinglysophospholipid acyltransferase activity; and (d) a nucleic acid thatcomprises a nucleotide sequence encoding a protein consisting of anamino acid sequence sharing an identity of 95% or more with the aminoacid sequence consisting of SEQ ID NO: 2 and having lysophospholipidacyltransferase activity.
 3. A cDNA or recombinant vector comprising anucleic acid of any one of (a)-(e) below: (a) a nucleic acid thatcomprises a nucleotide sequence encoding a protein consisting of anamino acid sequence with deletion, substitution or addition of one to 50amino acids in the amino acid sequence shown in SEQ ID NO: 2, and havingthe activity of increasing the proportion of arachidonic acid in thecompositional ratio of fatty acids in a host transformed with arecombinant vector containing the nucleic acid as compared with theproportion in the compositional ratio of fatty acids in a host that hasnot been transformed with the vector; (b) a nucleic acid that hybridizesunder hybridization conditions of 0.1×SSC-1×SSC at 60° C.-65° C. andwashing conditions of 0.2×SSC-2×SSC at 50° C.-68° C. to a nucleic acidconsisting of a nucleotide sequence full length complement to thenucleotide sequence consisting of SEQ ID NO: 1 and that comprises anucleotide sequence encoding a protein having the activity of increasingthe proportion of arachidonic acid in the compositional ratio of fattyacids in a host transformed with a recombinant vector containing thenucleic acid as compared with the proportion in the compositional ratioof fatty acids in a host that has not been transformed with the vector;(c) a nucleic acid that comprises a nucleotide sequence sharing anidentity of 90% or more with the nucleotide sequence consisting of SEQID NO: 1 and encoding a protein having the activity of increasing theproportion of arachidonic acid in the compositional ratio of fatty acidsin a host transformed with a recombinant vector containing the nucleicacid as compared with the proportion in the compositional ratio of fattyacids in a host that has not been transformed with the vector; (d) anucleic acid that comprises a nucleotide sequence encoding a proteinconsisting of an amino acid sequence sharing an identity of 90% or morewith the amino acid sequence consisting of SEQ ID NO: 2 and having theactivity of increasing the proportion of arachidonic acid in thecompositional ratio of fatty acids in a host transformed with arecombinant vector containing the nucleic acid as compared with theproportion in the compositional ratio of fatty acids in a host that hasnot been transformed with the vector; and (e) a nucleic acid thathybridizes under hybridization conditions of 0.1×SSC-1×SSC at 60° C.-65°C. and washing conditions of 0.2×SSC-2×SSC at 50° C.-68° C. to a nucleicacid consisting of a nucleotide sequence full length complement to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 and that comprises a nucleotide sequenceencoding a protein having the activity of increasing the proportion ofarachidonic acid in the compositional ratio of fatty acids in a hosttransformed with a recombinant vector containing the nucleic acid ascompared with the proportion in the compositional ratio of fatty acidsin a host that has not been transformed with the vector.
 4. The cDNA orrecombinant vector of claim 3, which is (a), (c), or (d) below: (a) anucleic acid that comprises a nucleotide sequence encoding a proteinconsisting of an amino acid sequence with deletion, substitution oraddition of 1-25 amino acids in the amino acid sequence shown in SEQ IDNO: 2, and having the activity of increasing the proportion ofarachidonic acid in the compositional ratio of fatty acids in a hosttransformed with a recombinant vector containing the nucleic acid ascompared with the proportion in the compositional ratio of fatty acidsin a host that has not been transformed with the vector; (c) a nucleicacid that comprises a nucleotide sequence sharing an identity of 95% ormore with the nucleotide sequence consisting of SEQ ID NO: 1 andencoding a protein having the activity of increasing the proportion ofarachidonic acid in the compositional ratio of fatty acids in a hosttransformed with a recombinant vector containing the nucleic acid ascompared with the proportion in the compositional ratio of fatty acidsin a host that has not been transformed with the vector; and (d) anucleic acid that comprises a nucleotide sequence encoding a proteinconsisting of an amino acid sequence sharing an identity of 95% or morewith the amino acid sequence consisting of SEQ ID NO: 2 and having theactivity of increasing the proportion of arachidonic acid in thecompositional ratio of fatty acids in a host transformed with arecombinant vector containing the nucleic acid as compared with theproportion in the compositional ratio of fatty acids in a host that hasnot been transformed with the vector.
 5. The cDNA or recombinant vectorof claim 1 wherein the encoded protein belongs to the membrane-boundO-acyltransferase family.
 6. A cDNA or recombinant vector comprising anucleic acid of any one of (a)-(d) below: (a) a nucleic acid thatcomprises the nucleotide sequence shown in SEQ ID NO: 1; (b) a nucleicacid that comprises a nucleotide sequence encoding a protein consistingof the amino acid sequence shown in SEQ ID NO: 2; (c) a nucleic acidthat comprises the nucleotide sequence shown in SEQ ID NO: 4; and (d) anucleic acid that comprises the nucleotide sequence shown in SEQ ID NO:5.
 7. An isolated protein of (a) or (b) below: (a) a protein consistingof an amino acid sequence with deletion, substitution or addition of oneto 50 amino acids in the amino acid sequence of SEQ ID NO: 2, and havinglysophospholipid acyltransferase activity; or (b) a protein consistingof an amino acid sequence sharing an identity of 90% or more with theamino acid sequence consisting of SEQ ID NO: 2 and havinglysophospholipid acyltransferase activity.
 8. The protein of claim 7,which is (a) or (b) below: (a) a protein consisting of an amino acidsequence with deletion, substitution or addition of 1-25 amino acids inthe amino acid sequence of SEQ ID NO: 2, and having lysophospholipidacyltransferase activity; or (b) a protein consisting of an amino acidsequence sharing an identity of 95% or more with the amino acid sequenceconsisting of SEQ ID NO: 2 and having lysophospholipid acyltransferaseactivity.
 9. An isolated protein of (a) or (b) below: (a) a proteinconsisting of an amino acid sequence with deletion, substitution oraddition of one to 50 amino acids in amino acid sequence of SEQ ID NO:2, and having the activity of increasing the proportion of arachidonicacid in the compositional ratio of fatty acids in a host transformedwith a recombinant vector containing a nucleic acid encoding the aminoacid sequence as compared with the proportion in the compositional ratioof fatty acids in a host that has not been transformed with the vector;or (b) a protein consisting of an amino acid sequence sharing anidentity of 90% or more with the amino acid sequence consisting of SEQID NO: 2 and having the activity of increasing the proportion ofarachidonic acid in the compositional ratio of fatty acids in a hosttransformed with a recombinant vector containing a nucleic acid encodingthe amino acid sequence as compared with the proportion in thecompositional ratio of fatty acids in a host that has not beentransformed with the vector.
 10. The protein of claim 9, which is (a) or(b) below: (a) a protein consisting of an amino acid sequence withdeletion, substitution or addition of 1-25 amino acids in the amino acidsequence of SEQ ID NO: 2, and having the activity of increasing theproportion of arachidonic acid in the compositional ratio of fatty acidsin a host transformed with a recombinant vector containing a nucleicacid encoding the amino acid sequence as compared with the proportion inthe compositional ratio of fatty acids in a host that has not beentransformed with the vector; or (b) a protein consisting of an aminoacid sequence sharing an identity of 95% or more with the amino acidsequence consisting of SEQ ID NO: 2 and having the activity ofincreasing the proportion of arachidonic acid in the compositional ratioof fatty acids in a host transformed with a recombinant vectorcontaining a nucleic acid encoding the amino acid sequence as comparedwith the proportion in the compositional ratio of fatty acids in a hostthat has not been transformed with the vector.
 11. The protein of claim7, which belongs to the membrane-bound O-acyltransferase family.
 12. Anisolated protein consisting of the amino acid sequence shown in SEQ IDNO:
 2. 13. An isolated cell transformed with the recombinant vector ofclaim
 1. 14. A fatty acid composition obtained by culturing thetransformed cell of claim 13 wherein the proportion of arachidonic acidin the compositional ratio of fatty acids in said fatty acid compositionis higher than the proportion of arachidonic acid in the fatty acidcomposition obtained by culturing a non-transformed host.
 15. A methodfor preparing a fatty acid composition, comprising collecting the fattyacid composition of claim 14 from cultures of a cell transformed cellwith a recombinant vector comprising a nucleic acid of any one of(a)-(e) below: (a) a nucleic acid that comprises a nucleotide sequenceencoding a protein consisting of an amino acid sequence with deletion,substitution or addition of one to 50 amino acids in the amino acidsequence shown in SEQ ID NO: 2, and having lysophospholipidacyltransferase activity; (b) a nucleic acid that hybridizes underhybridization conditions of 0.1×SSC-1×SSC at 60° C.-65° C. and washingconditions of 0.2×SSC-2×SSC at 50° C.-68° C. to a nucleic acidconsisting of a nucleotide sequence full length complement to thenucleotide sequence consisting of SEQ ID NO: 1 and that comprises anucleotide sequence encoding a protein having lysophospholipidacyltransferase activity; (c) a nucleic acid that comprises a nucleotidesequence sharing an identity of 90% or more with the nucleotide sequenceconsisting of SEQ ID NO: 1 and encoding a protein havinglysophospholipid acyltransferase activity; (d) a nucleic acid thatcomprises a nucleotide sequence encoding a protein consisting of anamino acid sequence sharing an identity of 90% or more with the aminoacid sequence consisting of SEQ ID NO: 2 and having lysophospholipidacyltransferase activity; and (e) a nucleic acid that hybridizes underhybridization conditions of 0.1×SSC-1×SSC at 60° C.-65° C. and washingconditions of 0.2×SSC-2×SSC at 50° C.-68° C. to a nucleic acidconsisting of a nucleotide sequence full length complement to anucleotide sequence encoding a protein consisting of the amino acidsequence shown in SEQ ID NO: 2 and that comprises a nucleotide sequenceencoding a protein having lysophospholipid acyltransferase activity. 16.A food product comprising the fatty acid composition of claim
 14. 17. Amethod for using a recombinant vector of claim 1 to increase theproportion of arachidonic acid in the compositional ratio of fatty acidsin an isolated host cell transformed with the vector as compared withthe proportion in the compositional ratio of fatty acids in an isolatedhost cell that has not been transformed with the vector, the methodcomprising: transforming the isolated host cell with the vector; andallowing the transformed host cell to produce arachidonic acid.
 18. AcDNA or recombinant vector comprising a nucleic acid of any one of(a)-(e) below: (a) a nucleic acid that comprises a nucleotide sequenceencoding a protein consisting of an amino acid sequence with deletion,substitution or addition of one to 50 amino acids in the amino acidsequence shown in SEQ ID NO: 2, and involved in the conversion from18:3(n-6)-PL to 18:3(n-6)-CoA or conversion from DGLA-CoA to DGLA-PL;(b) a nucleic acid that hybridizes under hybridization conditions of0.1×SSC-1×SSC at 60° C.-65° C. and washing conditions of 0.2×SSC-2×SSCat 50° C.-68° C. to a nucleic acid consisting of a full lengthcomplement to the nucleotide sequence consisting of SEQ ID NO: 1 andthat comprises a nucleotide sequence encoding a protein involved in theconversion from 18:3(n-6)-PL to 18:3(n-6)-CoA or conversion fromDGLA-CoA to DGLA-PL; (c) a nucleic acid that comprises a nucleotidesequence sharing an identity of 90% or more with the nucleotide sequenceconsisting of SEQ ID NO: 1 and encoding a protein involved in theconversion from 18:3(n-6)-PL to 18:3(n-6)-CoA or conversion fromDGLA-CoA to DGLA-PL; (d) a nucleic acid that comprises a nucleotidesequence encoding a protein consisting of an amino acid sequence sharingan identity of 90% or more with the amino acid sequence consisting ofSEQ ID NO: 2 and involved in the conversion from 18:3(n-6)-PL to18:3(n-6)-CoA or conversion from DGLA-CoA to DGLA-PL; and (e) a nucleicacid that hybridizes under hybridization conditions of 0.1×SSC-1×SSC at60° C.-65° C. and washing conditions of 0.2×SSC-2×SSC at 50° C.-68° C.to a nucleic acid consisting of a full length complement to a nucleotidesequence encoding a protein consisting of the amino acid sequence shownin SEQ ID NO: 2 and that comprises a nucleotide sequence encoding aprotein involved in the conversion from 18:3(n-6)-PL to 18:3(n-6)-CoA orconversion from DGLA-CoA to DGLA-PL.
 19. An isolated protein of (a) or(b) below: (a) a protein consisting of an amino acid sequence withdeletion, substitution or addition of 1-50 amino acids in the amino acidsequence of SEQ ID NO: 2, and involved in the conversion from18:3(n-6)-PL to 18:3(n-6)-CoA or conversion from DGLA-CoA to DGLA-PL; or(b) a protein consisting of an amino acid sequence sharing an identityof 90% or more with the amino acid sequence consisting of SEQ ID NO: 2and involved in the conversion from 18:3(n-6)-PL to 18:3(n-6)-CoA orconversion from DGLA-CoA to DGLA-PL.